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MICROBIOLOGY

PRACTICAL GUIDE (A)

2010

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PRACTICAL I Introduction Microorganisms, living creatures too small to be seen with the unaided eye, have a profound influence on daily life - often beneficial, sometimes harmful. - bread is made with the help of yeast that ferment sugar and produce carbon dioxide in the dough before it is baked ; - cheese, yogurt and sour cream require the presence of microorganisms that sour the milk from which they are made ; - some foods are treated to prevent microbial spoilage; - antibiotics cure such bacterial diseases as “step” turret bacterial pneumonias, gonorrhea and syphilis; - newborn children and adults are immunized against various diseases, including tetanus, diphtheria, whooping cough, measles and mumps, etc. - role in biogeochemical cycles The practicals deepen the knowledge of microorganisms; classical methods such as the use of aseptic techniques and enrichment culture based techniques; relationship between microorganisms and their environment; the basis of the phenotypical characterisationof microorganisms is similarly presented. The exercises are based on the investigation of representative strains of various bacterial groups. In designing the exercises, we carefully chose cultures and conditions that minimize the exposure of students to potentially pathogenic microorganisms and hazardous chemicals. Laboratory safety - wear a coat to protect your clothes - keep your desk free of unnecessary materials at all times and at the end of the period leave it free of all materials and equipment. - since many of the microorganisms with which you will be working are potentially pathogenic, it is imperative to develop aseptic techniques in handling and transferring them. - avoid any hand - to - mouth operations such as smoking, eating, drinking. - report immediately all accidents such as cuts, burns or spilled cultures to your instructor; take every precaution to avoid such accidents. - some of the chemicals employed in the laboratory can be hazardous if not handled properly, we have selected the experiments to minimize the use of such substances. A. Demonstration of microbes in the environment I. General Rules of sampling: samples always have to be representative! Recording details on site characteristics and other environmental factors will help when interpreting the sample results later on. Take extra care to avoid contaminating the sample container and the sample.

1. Sampling the air Air sampling in the context of microbiological assessment is the collection of airborne microorganisms. Some may impact product spoilage, product safety and human health. Collection of vegetative cells and spores may be achieved by passive or active methods. Passive methods usually involve settle plates (sedimentation) whereas active methods include

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impaction and impingement devices. The volume of air for sample collection depends on the device being used and on the anticipated concentration of the bioaerosol. Where low concentrations of microbial contaminants are expected, e.g. clean rooms, food production and operating theatres, impaction methods are generally chosen. In highly contaminated environments, impaction techniques may 'oversample' even over short timescales and impingement or filter samples are more appropriate. With the strict adherence to manufacturer's flow rates, sampling periods, culture media used and device placement, most techniques should yield comparable results, which are normally expressed in cfu/m³ (CFU= colony forming unit).

Fig. 1.1. Cascade impactor Soil sampling Soil tests measuremicrobial composition of different soil horizons. The accuracy of a soil test result is influenced by the laboratory analysis but may be influenced even more by the quality of the soil sample itself. Sample collection is extremely important in the accuracy and repeatability of a soil test. Sample handling following collection is also important. A soil sample which does not represent the area being sampled will be misleading. Soil physicochemical characteristics influence the level of biomass and the activity of microorganisms. Seasonal changes in soil moisture, soil temperature and C input from crop roots, rhizosphere products (i.e. root exudates, mucilage, sloughed cells, etc.), and crop residues can have a large effect on soil microbial biomass and its activity, which, in turn, affect the ability of soil to supply nutrients to plants through soil organic matter turnover. Water sampling (surface water) Take a labelled sterile sample bottle. Make sure you keep the lid on the bottle until you are ready to collect the sample.

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Hold the sterile bottle in one hand near the base, and then carefully remove and hold the cap with the other hand. Be careful not to touch the inside of the cap when sampling. Walk to a location at the water body, which is characteristic for the whole water (e.g. not polluted by sewage). Get down on one hand and knee beside the edge of the water body, then immerse the bottle downwards into the water with continuous motion and direction away from the body but parallel with the edge of the water body, to a depth of approximately 30 cm below the water surface and then continue to move the bottle in a horizontal motion until finally removing the bottle from the water body with the same continued action and motion when full. Tip enough of the water from the bottle to leave an air space of about 1-2 cm from the rim of the bottle. This air space is necessary to facilitate mixing of the sample in the laboratory. Carefully replace the cap immediately and tightly. Practice: B. Demonstration of microbes in the environment II. Demonstration of phototrophic microorganisms

plastic gum Air Aerobic zone Water (1-2 cm)

Sludge surface

Microaerophilic zone

\\\\\\\\\\\\\\\\ Non-sulfur photosynthetisers (rust red)

Anaerobic zone //////////////// "Purple" zone (anaerobic sulfur bacteria) Chromatium spp.

## # ### # ##

"Green-black" zone (anaerobic sulfur bacteria) Chlorobiaceae

2.5 cm sludge riched with CaSO4 and CaCO3 Fig.1.2. Winogradsky column for the enrichment of phototrophic bacteria The Winogradsky column is a small ecological system in which one can simulate microbial processes, for example, those occurring in the water and mud of a pond. The Winogradsky column is simply a clear container or cylinder packed with mud, shredded paper or powdered cellulose (as a source of energy and carbohydrate) salts of sulfate, carbonate and phosphate; and water. When sealed and exposed to light, a succession of microorganisms will evolve according to the amount of oxygen and light available at different points in the column. If the

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initial inoculum (the mud) contains the appropriate microbes, the column will enrich for photosynthetic bacteria, colorless sulfur bacteria and a range of other microorganisms. Results (after 6 weeks) At the end of the incubation period, use a Pasteur pipette to sample portions of the column at various levels. After removing a sample, place a drop on a slide and observe under microscope in a wet preparation. Some of the microorganisms you may find in your column are as follows: 1./ Aerobic, sulfide - oxidizing bacteria, for example Thiothrix and Beggiatoa. 2./ Sulfate-reducing microorganisms, such as Desulfovibrio. 3./ Sulfur-oxidizing bacteria that yield H2SO4 and thiosulfate, such as Thiobacillus. 4./ The red sulfide-oxidizing, anaerobic bacteria that grow in illuminated areas, for example Chromatium 5./ Anaerobic, sulfide-oxidizing bacteria that are all photosynthetic, such as Chlorobium. Upon microscopic examination, the purple sulfur bacteria can be recognized by their accumulation of intracellular sulfur granules, the nonsulfur and the green bacteria look much like spirilla. C. Demonstration of microbes in the environment III. Cultivation of microbes from our close environment (air, surfaces). Demonstration of the effect of hand-wash, etc. The aim of the present practical experiment is to demonstrate cultivable diversity from our surroundings and to demonstrate how inadequate cultivation methods may danger work in the laboratory.

1. Cultivation microbes from the air with sedimentation method: exposing media in Petri dishes to the air for different times (5’, 10’, 15’).

2. Testing surfaces of the laboratory (wall, floor, clothes, etc). 3. Testing human skin:

a. effect of hand washing Plates must be incubated at 28°C for one week. D. Enrichment of microorganisms Enrichment media are used to enrich (selectively grow) different microorganisms based on their specific characteristics (antibiotic resistance, diesel oil degradation capacity, heavy metal resistance) – selective enrichment of bacteria. Inoculate enrichment media with 1g of soil sample.

1. diesel-oil-containing medium. 2. Medium with cellulose as the sole carbon source . 3. Medium containing HgCl2 in high concentration.

After a week of incubation at 28°C, cultures must be spreaded to the adequate Petri dishes. E. Preparation of sauer kraut Sauerkraut is a product of fermentation. This completely changes the flavor of the raw material in addition to giving it long-term preservation qualities.

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The sauerkraut fermentation process utilizes the indigenous population of bacteria in the raw cabbage to produce lactic acid. This produces a low pH environment that allows few, if any, other bacteria to survive. The lactic acid is also what gives the kraut its characteristic sour flavor. Salt is added to the raw cabbage to draw out much of the water and to inhibit salt-intolerant bacteria. This allows acid-producing bacteria to get a strong foothold and dominate the population. The typical routine is to mix the cabbage with salt and then let it sit in a vat or barrel for the 6-week fermentation period. It is then ready to eat. It can be taken out as needed but that is were much of the trouble starts. Highly recommend processing it in a canner or pressure cooker to prevent further change, which is usually in the direction of spoilage or at least off-flavors. Mixed acid fermentation : Enterobacteriaceae Heterolactic fermentation: Leuconostoc mesenteroides Homolactic fermentation: Lactobacillus brevis, Lactobacillus plantarum The following simple procedure eliminates all the problems by advancing to the optimum stage.

o 2kgs shredded cabbage o 50 g salt (3 tablespoons)

Shred cabbage fine, put it in a large pan. Mix cabbage and salt with your hands. Pack jars gently with hands till is nearly full. Cover with clean rock or something heavy and close the bucket. During the curing process, kraut needs daily attention. Remove scum as it forms and wash and scald rock often to keep it free from scum and mould. At room temperature, fermentation will be complete in 10 to 12 days. Often there is not enough juice. If this happens, make thin brine by dissolving 30g of salt in 1L of water.

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PRACTICAL II

A. Preparation of microbiological culture media (preparing nutrient agar slants)

The survival and growth of microorganisms depend on available and a favorable

growth environment. Culture media are the nutrient solutions used in laboratories to grow microorganisms. For the successful culture of a given microorganism it is necessary to understand its nutritional requirements and then supply it with its essential nutrients in the proper form and proportions in a culture medium. The general composition of a medium is as follows:

1. H-donors and acceptors (approximately 1-15 g/L) 2. C-source (approximately 1-20 g/L) 3. N-source (approximately 0,2-2 g/L) 4. Inorganic nutrients e.g. S, P, (50mg/L) 5. Trace elements (0,1-1 µg/L) 6. Growth factors (aminoacids, purines, pyrimidines, occasionally 50 mg/L, vitamins

occasionally 0,1-1 mg/L) 7. Solidifying agent (e.g agar 10-20 g/L) 8. Solvent (usually distilled water) 9. Buffers

According to the consistency three types of media are used: liquid, or broth, media;

semisolid media; and solid media. The major difference among them is that solid and semisolid media contain a solidifying or gelling agent [such as agar, gelatine], whereas a liquid medium does not.

− Liquid media, such as nutrient broth, tryptic soy broth or glucose broth can be used in studies of growth and metabolism in which it is necessary to have homogenous media conditions, to follow optical density, and to allow early sampling for analysis of substrates and metabolic products. Tubes and flasks with liquid cultures can be incubated with either static or shaken incubation.

− Semisolid media can also be used in fermentation studies, in determining bacterial motility, and in promoting anaerobic growth.

− Solid media, such as nutrient agar, are used 1) for the surface growth of microorganisms in order to observe colony morphology, 2) for pure culture isolation, 3) often in the enumeration and isolation of bacteria from a mixed population by diluting the original bacteria suspension and spreading a small inoculum over the surface of the solidified medium and 4) to observe specific biochemical reactions (extracellular enzymes diffusing away from the colony can be detected as a result of their action on insoluble substrates present in the agar medium).

Solid media can be poured into either a test tube or Petri dish. If the medium in the test tube is allowed to harden in a slanted position, the tube is designated an agar slant; if the tube is allowed to harden in an upright position, the tube is designated an agar deep tube; and if the agar is poured into a Petri dish, the plate is designated an agar plate. Media are categorized by their composition:

− Chemically defined, or synthetic, media are composed of known quantity and quality of pure chemicals.

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− In routine bacteriology laboratory exercises, complex or nonsynthetic media are employed. These are composed of complex materials rich in vitamins and nutrients, the chemical composition of which is poorly defined. Three of the most commonly used components are beef extract, yeast extract and peptone (partially digested protein).

Media are categorized by their function: − An all-purpose medium, such as Tryptic Soy Agar, supports the growth of most

bacteria cultured in the laboratory. They do not contain any special additives. − Selective media enhance the growth of certain organisms while inhibiting the growth

of others due to the inclusion of particular substrate. − Differential media allow identification of microorganisms usually through the

(visible) physiological reactions unique to those bacteria. The most practical media are those that both select for and differentiate common pathogens.

− Enrichment media allow metabolically fastidious microorganisms to grow because of the addition of specific growth factors. Enrichment culture is one obtained with the use of selected media and incubation conditions to isolate the desired microorganisms from natural samples.

The preparation of media from commercially available dehydrated products is simple

and straightforward. Each bottle of dehydrated medium has instructions for preparation of its label. For example, to prepare a liter of tryptic soy broth, suspend 30 g of the dehydrated medium in 1.000 ml distilled water. Mix thoroughly in a 2 liter Erlenmeyer flask [always use a flask that holds twice the volume of media you are preparing]. Dispense and sterilize for 20 minutes at 121 0C [15 lbs pressure]. As noted, the amount of powder for 1.000 ml of water will be indicated. In case of preparation of media from a formula pons 500 ml of distilled water into a 2000ml Erlenmeyer flask. Then measure adequate amount of media components and dissolve completely in the water in the order of the formula. At the end rins the flask with the remaining 500 ml water. Mix the medium thoroughly, adjust the pH and sterilize.

If the medium lacks agar, the powder will usually dissolve without heating. If it contains agar, it is necessary to heat the medium until it starts to boil or even longer in order to completely dissolve the agar. Most of the exercises you will be doing in this manual will involve the use of sterile media culture tubes. These tubes must be capped in order to maintain media sterility. This can be accomplished by using cotton plugs, plastic foam plugs or plastic or metal caps. All of these caps keep cultures free of contamination while allowing air into the culture tube, and minimizing evaporation at the same time. It is sometimes desirable to use screw cap culture tubes. This is especially true when the culture, such as in the case of slants, may be sealed and stored for long periods. Culture broth can be dispensed with the pipetting machine, an automatic syringe, or a regular pipette. Agar deep tubes can be stored after sterilization for use in the preparation of Petri plates when needed. Some agar deeps may be stored at room temperature for several days before use. If longer periods of storage are required, they should be placed in the refrigerator in order to prevent drying of the agar. When Petri plates are needed, the agar deeps are melted either in a boiling water bath or by bringing them to 121 0C in an autoclave for 30 to 60 seconds, and then releasing the steam under slow exhaust. After the agar has melted, the pours are transferred to a 48 to 50 0C water bath and kept there for at least 5 to 10 minutes before use. The agar deeps should be cooled to about 50 0C before they are used to minimize the amount of steam condensation on the Petri plate lids after the agar has been poured. Agar does not solidify until its temperature drops to about 42 0C. When the deeps have reached 50°C , one is taken from the bath and the outside is dried with a paper towel. Its cap is removed and the top

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is briefly flamed using a Bunsen burner. The agar is immediately poured into a sterile Petri plate while holding the top carefully above the Petri plate bottom in order to avoid contamination. Replace the top, allow the agar to cool and harden, and store the Petri plates in an inverted position. B. Sterilization of media and equipment

Sterilization is the process of rendering a medium or material free of all forms of life. There are three basic ways in which sterilization of media and supplies can be achieved. The most useful approach is autoclaving, in which items are sterilized by exposure to steam at 121 0C and 15 for 15 minutes or longer, depending on the nature of the item. Under these conditions, microorganisms, even endospores, will not survive longer than about 12 to 13 minutes. Modern autoclaves are designed to ensure that all of the air has been expelled and only steam is present in the autoclave chamber. They are carefully temperature controlled as well. Almost all media and anything else that will resist 121 0C and steam can be sterilized this way.

Often dry glassware, such as pipettes and Petri plates, must be sterilized. Steam tends to etch glassware and also leaves it damp. Therefore, such items are generally dry - heat sterilized. The glassware is placed in an electric oven set to operate between 160 and 170 0C. Since dry heat is not as effective as wet heat, glassware must be kept at this temperature for about 2 hours or longer. Oven temperature must not rise above 180 0C or any cotton or paper present will char.

Sometimes media must be made from components that will not withstand heating at 121 0C. Such media can be sterilized by passing it through a (bacteriological) filter, which physically removes bacteria and larger microorganisms from the solution and thereby sterilizes them without heat. Scintered glass filters with ultrafine fitted disks [0.9 to 1.4 µm pore size] and Seitz asbestos - pad filter funnels [3 mm thick with 0.1 µm pores] are both quite effective sterilizing solutions. The most useful and popular approach by far is the use of specially prepared sterile, cellulose- or polycarbonate, etc.-based membranes of the appropriate pore size. Generally, membranes with 0.22 µm pores are employed in sterilization. A large number of different devices are commercially available for membrane sterilization of both large and small volumes. For example, one can use a filter flask with vacuum or syringe with positive pressure to force liquid through a special membrane filter holder.

Exercise:

a. Measure solid components, dissolve b. Make final concentration c. Adjust pH at solidification temperature (in hot form!) d. Sterilize e.

Nutrient agar: - meat extract 3g - peptone 5g - agar 18g - distilled water 1000ml pH. 7.0, Autoclave for 15’ at 121°C 1. With different equipments the pressure of vapor, temperature and the impact of time can be checked permanently.

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2. Chemical indicators shows wether the temperature and the time period was enough for sterilization. 3. The use of biological indicators are the most trustworthy methods to calibrate sterilization equipments. To it standard bacterial spore-preparatios are used (like Bacillus stearothermophilus). If the efficacy of starilization is insufficient, the test-microbe survives. Exercise: Controlling the efficacy of sterilization with use of sporepreparation of Bacillus stearothermophilus strain ATCC 7953. C. Demonstration of microbes in the environment Isolation of bacteria, evaluation the CFU (Colony Forming Unit) values from an environmental sample. Introduction to cultivation, nutrition and other growth conditions Viable germ count: the number of viable “germs” in a culture can be ascertained by determining the number of colony forming units CFU with the colony counting technique. (Sometimes microbes clump. A colony may grow from several microbesclustered together. For this reason, microbiologists often say colonies develop from CFU’s rather than from a single cell.) Between 20 and 200 CFU can be counted on a typical Petri plate. Microbial cultures of high density must be diluted before they are plated. A known volume [0,1ml] of these dilutions is plated onto suitable growth medium in the Petri dish. After the plates have been incubated up to a week, the average number of colonies on plates is determined.

The number of viable microbes per milliliter (or g) of the initial culture (sample) can be calculated from the average CFU’s and the known dilution factor. The viable count is invariably less than the total cell count because it measures only cells capable of dividing. The major disadvantages of the colony plating technique are : i./ the incubation period is lengthy, ii./ sterile media , pipets and plates are required , iii./ sampling and dilution errors occur. Two methods of this examinations are differentiated: 1) In the pour–plate method, a sample from an accurate dilution of microbes/sample is pipetted onto a Petri - dish, then agar medium is poured over the liquid and mixed. 2) In the spread–plate method, generally 0.1ml of the diluted sample is pipetted onto the surface of a solidified agar medium and spread with a sterilized, bent, glass rod.

The theory behind the technique of CFU establishes that a single microbe can grow and become a colony via division. These colonies are clearly different from each other, both microscopically and macroscopically. This technique allows the user to know how many CFU’s are present per mL in the sample. Therefore, it enables us to know the microbiological load and the magnitude of the infection in humans and animals, or the degree of contamination in samples of water, vegetables, soil or fruits and in industrial products and the equipment.

Quantifying Bacteria by Spread Plate

The number of bacteria in a solution can be readily quantified by using the spread plate technique. In this technique, the sample is appropriately diluted and a small aliquot is transferred to an agar plate. The bacteria are then distributed evenly over the surface by a special streaking technique. After colonies are grown, they are counted and the number of bacteria in the original sample is calculated.

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The end point of our analysis is the number of colony forming units per mL (CFU/mL) since we are counting the number of colonies rather than the actual number of bacteria. CFU/mL is actually a more useful determination than counting all the bacteria under a microscope, because in many bacterial populations, a significant number will be dead cells and thus of no interest.

Diluting the bacteria. Bacteria commonly grow up to densities around 109 CFU/mL, although the maximum densities vary tremendously depending on the species of bacteria and the media they are growing in. Therefore, to get readily countable numbers of bacteria, we have to make a wide range of dilutions and assay all of them with the goal of having one or two dilutions with countable numbers. We do this by making serial 10-fold dilutions of the bacteria that cover the entire probable range of concentrations. Then we transfer 0.1 mL of each dilution to an agar plate, which in effect makes another 10-fold dilution, since the final unit is CFU/mL and we only streak 0.1 mL.

Inoculating the plate. Streaking in this technique is done using a bent glass rod. 0.1 mL of bacterial suspension is placed in the center of the plate using a sterile pipette. The glass rod is sterilized by first dipping it into a 70% alcohol solution and then passing it quickly through the Bunsen burner flame. The burning alcohol sterilizes the rod at a cooler temperature than holding the rod in the burner flame, thus reducing the chance of you burning your fingers. When all the alcohol has burned off and the rod has air-cooled, streak the rod back and forth across the plate working up and down several times. Unlike streaking for isolation, you want to backtrack many times in order to distribute the bacteria as evenly as possible. Turn the plate 90 degrees and repeat the side to side, up and down streaking. Turn the plate 45 degrees and streak a third time. Do not sterilize the glass rod between plate turnings. Cover the plate and wait several minutes before turning it upside down for incubation. This will allow the broth to soak into the plate so the bacteria won't drip onto the plate lid.

Counting bacteria. Colonies are most readily counted using a plate counter. The plate counter has a light source and a magnifying glass making colonies easier to see. If at all possible, you don't want to count plates with more than 300 or less than 30 colonies. In the former case, the colonies , run together, and, in the latter, there are too few to allow statistically accurate counts. Once you count the colonies, multiply by the appropriate dilution factor to determine the number of CFU/mL in the original sample.

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CFU/mL or g: number of colonies on plate x 1/dilution rate

Fig. 2.1. An example of serial dilution and plating

Fig. 2.2. Usage of spreading rod Exercise: Make a serial dilution from 1g of soil or 1ml of water sample. Spread onto plates from each dilution. Thereafter, incubate at 28°C for one week. Evaluation of data on the next week. D. Evaluation of the results of enrichment technique. E. Measure the pH of sauerkraut F. Evaluation of the results of “Demonstration of microbes in the environment I.”

1g soil

99 mL

0.1 mL

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PRACTICAL III.

A. Isolation of microbes

Selected colonies will be transferred from Petri dishes into new media to tubes. B. Colony morphology

Microbes grow on solid media as colonies. A colony is defined as a visible mass of microorganisms all originating from a single mother cell, therefore a colony constitutes a clone of bacteria all genetically alike.

Observing colony morphology on inoculated plates On a given medium, a colony’s shape, color, consistency, surface appearance and size - for a given incubation time - are often characteristic, and these are often of use in the identification of particular bacterial strains. The full description of a colony can be very detailed. Thus e.g. the elevation of a colony may be flat, low convex, domed unbonate etc., its edge maybe entire [circular or unbroken], crenate [scalloped], lobed or fimbriate; its texture may be butyrous friable or mucoid; its surface may be matt or glossy; it may be whitish or pigmented or it may contain a dye taken up from the medium, or it may release water soluble pigment into the medium. The colonies of certain bacteria e.g. Bacillus can migrate across the surface of a culture plate, the tract of such movement is often marked by lines of bacterial growth which arise from the cells left behind by the migration colony. An interesting feature of certain bacterial colonies is the so-called smooth-rough variation. In many types of bacteria, some type of S-R variation is responsible for a change in the cell-surface composition, which occurs spontaneously during in vitro or in vivo growth. S-R variation was first recorded in enterobacteria, in which smooth [glossy] colony may be formed on primary isolation, and rough [dull] colonies may develop on subcultures.

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Fig. 3.1. Basic colony morphology types

Identify the following colonial characteristics/culture characteristics:

Whole shape of colony, size of colony (measure with a millimeter ruler), less than 1mm = punctiform (pin-point). EDGE/MARGIN OF COLONY: magnified edge shape CHROMOGENESIS (pigmentation): white, buff, red, purple, etc. Some pigments are water-soluble, others are not. If you take a large inoculum and place it in a tube of water or saline, do you see color? Do you see any pigment if the organism is growing in a broth medium? OPACITY OF COLONY: transparent (clear), opaque, translucent (almost clear, but distorted vision–like looking through frosted glass), iridescent (changing colors in reflected light) ELEVATION OF COLONY SURFACE OF COLONY: smooth, glistening, rough, dull (opposite of glistening), rugose (wrinkled) CONSISTENCY: butyrous (buttery), viscid (sticks to loop, hard to get off), brittle/friable (dry, breaks apart), mucoid EMULSIFIABILITY OF COLONY: Is it easy or difficult to emulsify? Does it form a uniform suspension, a granular suspension, or does not emulsify at all? ODOR: Absent or present? If it has an odor, what does it smell like?

C. Cultivation of microorganisms, transfer ofmicroorganisms, storage of strains

The practice of transferring microbes from one medium to a new sterile medium is done as shown on Figure 3.3. in the Practical Instruction Guide. The proper way of sterilizing the inoculating loop is presented in Figure 3.2. in the Practical Instruction Guide.

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It is important to keep a Bunsen-burner on throughout the process to prevent contamination from the air. Always mark each new test-tube properly before transferring microorganisms (date, sign of the given strain). Never put the caps or cotton plugs down on laboratory surfaces, keep them in your hand throughout the process.

Fig 3. 2. The proper way of sterilizing the inoculating loop

Fig. 3.3. The practice of transferring microbes from one medium to a new sterile

medium

or or or

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D. Preparation of pure cultures Streak plate method

Streaks are made across the surface of the agar with a loop full (inoculating loop) of mixed culture [suspension of Staphylococcus aureus and E. coli or Serratia marcescens and Micrococcus luteus]. There are reversal different methods for streaking, but in all methods the objective is to deposit most of the organisms in the first few streaks. Thus, as one continues to streak the loop back and forth from one section to another of the Petri dish, fewer bacteria will remain on the loop. If done properly, the last streaks should leave individual bacteria appeare sufficiently apart from each other so that, after growth, the colonies that have developed from individual bacteria will be well-separated from each other. A single colony then can be transferred to a sterile medium, and a pure culture [axenic] will grow. Strains: Mixed suspension of type strains Culture medium: Nutrient agar plates Equipment, material: sterile water, pipette, inoculating loop

Fig. 3.4. Preparation of pure cultures

E. Application of REPLICA-technique for the isolation of antibiotic-sensitive or -resistant microbes

Using the replica technique, we are preparing “copies” from the discrete bacterial colonies that grew on the surface of a Petri-plate. The velvet should be strung onto a wooden, plastic or metal log. The Petri-dish is then carefully pressed against the surface of the velvet. The fine filaments of the velvet function as an inoculating loop/needle so that we are able to transfer all the colonies from the agar plate onto new, sterile agar plates in just one step. Using selective media (e.g. containing a specific antibiotic) we can get special metabolic mutants, microbes of distinct abilities. We are creating replicates from agar plates infected last week (spread plate from soil and water environmental samples) to agar plates containing a certain antibiotic. Be careful

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to “replicate” the plates at the exact same position as the original agar plates. The incubation of the replicate plates is at 28oC. Evaluation of the results happens the following week with the absence or presence of the colonies present on the original plates.

Equipment, material: sterile velvet on a replica log, agar plates containing different antibiotics

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PRACTICAL IV A.The transfer of the unidentified strain to fresh media (2 copies): (Everybody gets unidentified bacteria to work with throughout the practical course) Strains: Own unindentified strain Culture medium: Nutrient agar slants Equipment, material: Inoculating loop B.Demonstration and application of different anaerobic culturing techniques B/1. Deep agar tube containing sodium-thioglycollate

The oldest and most common method of the cultivation of anaerobic bacteria is culturing them deep in reduced semisolid or solid media. The most important property of these nutrient media is the suitably low (Eh<-60mV) redox potential. Different indicators within the culture media serve to test the appropriately low redox potential. These are dyes that are colorless in a reduced environment. The most suitable indicators are: resazurin and methylene blue, which show color only when in an oxidized environment (where resazurin is red and methylene blue is blue). To ensure that oxygen does not get into the culture media either, the inoculated media is sealed during incubation (melted and heat sterilized vaseline, or vaseline-paraffine 1-1 mixture is layered on top of the culture media). Semi-solid agar deep tubes are prepared with a small amount of agar, so that these media are appropriate for the cultivation of anaerobes without sealing, since agar itself can reduce convection currents, thus reoxidation as well and agarcolloid applied in small concentration are reductive. To further lower the redox potential in semisolid media, reductive compounds could be used (sodium-thioglycollate and cystein). Exercise: Add 1 ml of sediment suspension or water sample to sodium-thioglycollate containing melted agar deep tubes. Incubate the tubes at 28°C for a week. Following incubation, the amount of colonies formed inside/within the media should be used to estimate the anaerobe count of the original sample.

Sample: soil Culture medium: Sodium-thioglycollate agar deep tube Equipment, material: sterile pipette, sterile distilled water B/2. Cultivation of anaerobic bacteria on Marino-plates

The appropriate method of the cultivation of anaerobes should be chosen with consideration of the sensitivity of the given organism for the remaining oxygen concentration within the media or the surrounding atmosphere. For the cultivation of bacteria that are sensitive to even trace amounts of oxygen (e.g. methanogens and sulphate reducing bacteria), the best technique is the use of an anaerobic system, filled with an appropriate gas, contacting the outside world through a sluice gate that removes oxygen entering the system with the use of a catalyst. Those microbes that are not so sensitive to trace amounts of oxygen and are able to survive temporarily high oxygen concentrations (e.g. sampling) by forming endospores, can be cultivated in anaerobic jars or inside usually semisolid media also containing special reductive agents but incubating at normal conditions. The problem is that colonies forming inside “anaerobic” agar deep tubes are difficult to examine and isolate. This problem can be

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solved by using Marino-plates (better known as Brewer-plates), a combination of a Petri-dish and “anaerobic” agar. The Marino-plates provide a bigger surface area to examine the colony-forming properties of anaerobic bacteria and also make the isolation of such bacteria a lot easier. Exercise: Prepare a 6-member dilution series (one in ten). If the cultivation of Clostridia is the aim of the practical, the test tubes containing the dilution series should be put into an 80°C water-bath for 10 minutes to select for endospore forming organisms. The glass Petri-dishes should be unwrapped under a laminar box. Pipette 100 µl from a given dilution onto the inside surface of the Petri-dish top. Then pour culture media (that has cooled down to approximately 45°C) into the Petri-dish and thoroughly mix the diluted sample with the nutrient media. Put the bottom of the Petri-dish (with a smaller diameter than the top) onto the top before the media solidifies; thus creating a thin culture media between the bottom and the top of the Petri-dish. The Marino-plates then should be wrapped into foil again and incubated for at least 1-2 days, maybe even for a week depending on the results. Following incubation, observe the plates where intense bacterial growth can be detected. Examine the individual colonies under a stereo microscope and try to isolate some of the colonies by making stab cultures of agar deep tubes.

Equipment, material: sterile Petri-dishes wrapped into foil, warm semisolid “anaerobic” agar (e.g. differential RMAC for the cultivation of Clostridia), some sample taken from anaerobic environment (e.g. an anaerobe layer of lake sediment), 80°C water-bath, a series of test tubes containing 9 ml sterile distilled water for preparing a serial dilution. B/3. Cultivation of Clostridia in GasPak anaerobic system

The GasPak® anaerobic system is a jar that is usually transparent and can be sealed; contains a palladium catalyst and a disposable H2+CO2 generator and redox indicator. Cultures are placed into the jar along with an envelope containing chemicals that release hydrogen and carbon dioxide when activated by water. The envelope includes two tablets, one that contains NaBH4 that generates hydrogen when reacting with water; the other tablet contains citric acid and sodium-hydrogen-carbonate that generates CO2 when coming in contact with water. The CO2 contributes to the support of growth of fastidious anaerobes. Water is added to the envelope; the jar is then sealed. In the presence of the palladium catalyst, hydrogen reacts with oxygen to form water. The reaction removes free oxygen from the inside of the jar.

Equipment, material: anaerobic jar, sterile pipette, spreading glass rod, bismuth-sulfite agar medium Exercise: Put the inoculated Petri-dishes with the inoculated surface facing down into the anaerobic jar (Figure 4.1). Open the redox indicator and place it next to the plates, so that the indicator strip can be seen from the outside (the indicator strip will turn blue within seconds). Cut the gas generator envelope with scissors, and pipette 10 ml water through the hole. Close the jar and screw on the clamp, then put the jar into the thermostat.

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Figure 4.1 The anaerobic jar system

B/4. Estimation of sulphate reducing bacterial count by MPN method

If sulphate ions are present in the anaerobic environment, the fermentation end product can be further transformed by the involvement of sulphate-reducing bacteria (SRB). The SRB is such a specialized group of bacteria whose members proliferate under anaerobic conditions, and use the energy needed for growth by oxidizing various organic substances with sulphate, which is then used as the final electron acceptor during anaerobic respiration. The final product of this respiration is the sulphide ion or hydrogen sulphide (H2S). SRB are more “restricted” than fermentative microorganisms in their spectrum of utilized organic compounds. Most of the fermentative microorganisms are able to transform very complex organic compounds and polymers while the substrates for SRB are mainly different low molecular weight organic compounds, the final products of acetogenic fermentation (eg. lactate, acetate, proprionate). So the SRB are dependent on acid producing bacteria that provide electron donors to the them .

The SRB is a very diverse group. According to the 16S rDNA sequence analysis, the SRB seems to be a polyphyletic group. The SRB genera form four main groups: the Gram-negative mesophile SRB; the Gram-positive endospore forming SRB; thermophile eubacteria and the thermophile archea SRB. All four groups utilize sulphate as the terminal electron acceptor during anaerobic respiration.

SRB can be divided into two bigger groups by their metabolic properties. The members of the first group can oxidize the different organic substrates to acetate (“incomplete oxidation” the genera of Desulfovibrio, Desulfotomaculum, Desulfobulbus), while members of the other group are capable to oxidize these substrates completely to carbon dioxide (the genera of Desulfobacter, Desulfococcus, Desulfosarcina). Some members of both groups show hydrogenase activity, thus are able to use hydrogen as electron donor.

Sulphate-reducing bacteria have economic importance since they play an important role in metal corrosion (their activity can cause the corrosion of gasoline or gas pipes on the inside)

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and in wastewater treatment processes (part of the biofilm-forming community). Therefore it is important to study their distribution within a sample.

Equipment, material: the upper 2-5 cm of lake sediment, sterile spoon, sterile 100 ml Erlenmeyer flask, sterile small test tubes, sterile pipette, well micro-titer plates, PMB broth, anaerobic chamber The composition of the PMR medium: Generalbasal medium:

KH2 PO4 0,5 g NH4Cl 1,0 g CaSO4 1,0 g MgSO4x7H2O 2,0 g Yeast extract 1,0 g Ca-Na-lactate 4,0 g Distilled water 980 ml

pH: 7,0-7,5, sterilization: 1 atm, 121°C, 15 min

FeSO4 FeSO4x7H2O 1,1 g Distilled water 10 ml

Reductive solution:

Na-thioglycollate 0,1 g Ascorbic acid 0,1 g Distilled water 10 ml

The preparation of FeSO4 and reductive solutions is done inside the anaerobic chamber. These solutions are sterilized by filtration and added to the autoclaved basal medium of approximately 60°C. Exercise: To estimate the bacterial count of the SRB, the culture dependent Most-Probable-Number (MPN) method is applied. The differential broth Postgate’s Medium B (PMB) is used, which is suitable for the cultivation of the two most common genera of SRB: the Desulfovibrio and the Desulfotomaculum. It is easy to detect the presence and activity of SRB in the PMB broth, since it contains Fe ions that form black Fe-sulphide precipitate with the end product of sulphate reduction.

Prepare a 6-member dilution series as follows: Measure 3,0 ml (or 3,0 g) of sample into a 27 ml PMB broth containing 50 ml Erlenmeyer flask. Homogenize the solution with a vortex at 22°C for 5-10 minutes. Pipette 0,3 ml of the obtained suspension into a test tube containing 2,7 ml PMB broth. Mix vigorously, then transfer 0,3 ml suspension again into a new test tube, and so on. From each dilution (including the original sample), pipette 0,3 ml onto a 96-well microtiter plate in five parallels (5 test-tube MPN method). The incubation of the microtiter plates takes place inside the anaerobic chamber of Forma Scientific at 30°C for 2 weeks. The enumeration of total SRB count is estimated by the color change of the PMB nutrient broth after 1-2 weeks (black precipitate of iron-sulphide). The SRB count is determined by counting the positive (black colored) wells of the plate and transforming it to the appropriate McCardy characteristic number to obtainthe MPN value.

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B/5. Demonstration of the anaerobic chamber (glove-box)

The anaerobic chamber is a device suitable for the cultivation of strictly anaerobic bacteria. Inside the system, anoxic conditions can be maintained while being able to perform microbiological operations (microscopy, isolation, inoculation, etc.). Oxygen is removed from the system with vacuum, then a gas mixture (10% H2, 10% CO2, 80% N2) is introduced into the system with a slightly positive pressure. The detection of trace amounts of oxygen is preformed by methylene blue or resazurin redox indicators and the elimination of such oxygen is completed by a palladium catalyst. Active carbon inside the anaerobic chamber serves to bind catalyst poisons (e.g. H2S) and other substances that are toxic for bacterial cells.

C. Preparation of pure cultures II.

Practical C: The purified strains from last week’s practical should be isolated onto fresh, sterile agar slants.

D. Checking the pH of the sour-cabbage

E. Evaluation of the replica plates from Practical III.

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PRACTICAL V

A.The transfer of the unidentified strain to fresh media (2 copies): Strains: Own unindentified strain Culture medium: Nutrient agar slants Equipment, material: Inoculating loop DEMONSTRATION OF THE EFFECT ENVIRONMENTAL FACTORS ON BACTERIAL GROWTH

Microbes generally adapt easily to the their natural habitats, however the change in the physico chemical properties of their environment (e.g. temperature, pH, light, oxygen tension, osmotic pressure) could greatly affect their life function (e.g. growth rate, pigmentation, endospore formation). B.Temperature B/1. Bacterial growth at different temperatures Exercise: Transfer the different strains of bacteria (from suspension or agar slants) onto fresh nutrient agar slants and incubate each strain at 4°C, 28°C and 37°C. Observe the rate of bacterial growth on the medium after a week-long incubation.

Strains: Own unidentified strain B/2. Temperature tolerance Exercise: Place the already sterilized nutrient medium prepared in big test tubes into water bath of 60°C and 95°C. Pipette 0,1-0,1 ml bacterial suspension aseptically into the test tubes, mix the liquid media with the suspension and incubate at the given temperature for 10 minutes, then pour the contents of each test tube into sterile Petri-dishes. Observe bacterial growth, then estimate the temperature tolerance of each strain by the number of colonies formed on the agar plates following incubation at 28°C for a week.

Strains: E. coli suspension Bacillus subtilis suspension

Materials: sterile pipette and tips, nutrient agar, water-bath (60°C and 95°C) C. Effect of pH on microbial growth

Besides temperature, the hydrogen ion concentration of an organism’s environment exerts the greatest influence on its growth. The concentration of hydrogen ions, which is customarily designated by the term pH (-log[H+]), effects transport through the cell membrane and limits the activity of enzymes. As in the case of temperature, an optimum (concentration of hydrogen ions in which the bacterial growth is most intense) exists for each organism. Minimum and maximum hydrogen ion concentrations are pH values where an organism still shows growth. These values are realistic only when other environmental factors remain constant. If the composition of the medium, incubation temperature, or osmotic pressure is changed, the pH requirements become different.

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Exercise: Pipette 0,1-0,1 ml bacterial suspension aseptically into test tubes containing nutrient broth of different pH values (pH=5, pH=7, pH=9). Estimate the pH tolerance of each strain by examining the growth rate of the bacteria following incubation at 28°C for a week (observe the turbidity of each broth and compare it with a blank test tube that has not been inoculated with bacteria).

Strains: E. coli Staphylococcus aureus

Materials: sterile culture media, inoculating loop, sterile water in test tubes D. The effect of UV radiation on bacterial growth

Most microorganisms are very sensitive to UV radiation. The UV range covers a wide band of the electromagnetic spectrum (4nm-400nm) but only a narrow range of this spectrum is responsible for the germicidal effect. There is a very strong bactericidal effect at the wavelength of 265 nm, due to the damage caused within the DNA replication mechanism. Usually pigmented and endospore-froming bacteria are most resistant to the effects of ultraviolet radiation.

Exercise: Expose infected agar plates to UV radiation for different time intervals (10-20-30 minutes). The germicidal effect should be evaluated following incubation at 28°C for a week.

Strains: Own unindentified strain Materials: sterile nutrient plates, sterile pipette and tips, glass rod, sterile water in test tubes

E.Osmotolerance of microorganisms, effect of water activity (aw) on microbial growth Water activity, (aw). Qualitatively, aw is a measure of unbound, free water in a system, available to support biological and chemical reactions. Water activity affects microorganisms' survival and reproduction, enzymes, and chemical reactions. The water activity of a substance is quantitatively equal to the vapor pressure of the substance divided by the vapor pressure of pure water (both measured at the same temperature). Water molecules are loosely oriented in pure liquid water and can easily rearrange. When other substances (solutes) are added to water, water molecules orient themselves on the surface of the solute and the properties of the solution change dramatically. The microbial cell must compete with solute molecules for free water molecules. aw varies very little with temperature over the range of temperatures that support microbial growth. A solution of pure water has an aw of 1.00. The addition of solute decreases the aw to less than 1.00.

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Water Activity of Various NaCl Solutions

Percent NaCl (w/v) Water Activity (aw)

0.9 0.995

3.5 0.98

7.0 0.96

10.0 0.94

16.0 0.90

22.0 0.86

The aw of a solution may dramatically affect the ability of heat to kill a microbe at a given temperature. For example, a population of Salmonella typhimurium is reduced tenfold in 0.18 minutes at 60°C if the aw of the suspending medium is 0.995. If the aw is lowered to 0.94, 4.3 min are required at 60°C to cause the same tenfold reduction. An aw value stated for a microbr is generally the minimum aw which supports growth. At the minimum aw, growth is usually minimal, increasing as the aw increases. At aw values below the minimum for growth, microbes do not necessarily die, although some proportion of the population does die. The bacteria may remain dormant, but infectious. Most importantly, aw is only one factor, and other factors (e.g., pH, temperature) must be considered. It is the interaction between factors that ultimately determines if a microbe will grow or not. A number of food spoilage microbes can grow within the range 0.8 - 0.6.

Microbe Water Activity (aw)

Bacteria in general 0.91

Halophiles 0.75

Yeasts in general 0.88

Xerotrophic yeasts < 0.80

Molds in general 0.80

Xerotrophic molds 0.70

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The aim of the practical is to compare the osmotolerance of bacteria originating from diverse environments. The lowering of aw is achieved with the addition of NaCl to the culture media. Exercise: Inoculate bacterial suspension into culture broth of different salt concentration values (0%, 5%, 10%). The germicidal effect should be evaluated following incubation at 28°C for a week. Estimate the osmotolerance of each strain by examining the growth rate of the bacteria following incubation at 28°C for a week (observe the turbidity of each broth and compare it with a blank test tube that has not been inoculated with bacteria).

Strains: E. coli Staphylococcus aureus

Materials: sterile culture broth, inoculating loop F. Demonstration of the efficacy of different disinfectants Microbiological laboratories in epidemiological work, medical facilities or the industry (pharmaceutical companies, food) use disinfectant solutions for preventive, continuous or terminal disinfection. In the case of an actual epidemiological event (epidemic, accumulation of contagion), the effectiveness of these disinfectants is randomly inspected. The principle of efficacy testing is that the disinfectant in question is mixed with the suspension of the test bacterium, then, after a defined time interval, the disinfectant-microbe suspension is sampled with an inoculating loop and spread onto the surface of suitable nutrient media. Following the allotted incubation period, conclusions can be made whether the disinfectant, within the exposure time interval, killed the test microbe by observing the growth of the test microbe or the absence of microbial growth. Exercise: Measure 9-9 ml of each previously diluted disinfectant solution and the control solution into sterile test tubes and place the test tubes into a water-bath of 25°C. Place a sterile glass rod into the suspension of the test microbe (18-24 h) for 10 minutes. Following the 10-minute incubation, take out the glass rod, let the excess suspension flow down the side of the test tubes. Now place the “infected object” into the appropriate disinfectant solution and leave it there for the fixed incubation period of 1, 5, 15, 30, 45 or 60 minutes, then take it out, immerse in sterile water for 1 minute (to remove the remaining disinfectant from the glass rod). Inoculate TSA plates with the glass rod and store the inoculated plates at 28°C for a week. Evaluate bacterial growth compared with the control that has not been inoculated on a scale of five (-, ±, +, ++, +++).

Strains: Staphylococcus aureus Pseudomonas aeruginosa Bacillus subtilis

Materials: 1% and 2% Na-hypochlorite-solutions, other disinfectant solutions Control solution: 0,9% NaCl solution Equipments: pipette and tips, vortex, inoculating loop, water-bath, TSA plates

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No plates Inspected material Inoculated

microbe 1’ 5’ 15’ 30’ 45’ 60’

1. 1% Na-hypochlorite Staphylococcus aureus 2. 1% Na-hypochlorite Pseudomonas aerug. 3. 1% Na-hypochlorite Bacillus subtilis 4. 2% Na-hypochlorite Staphylococcus aureus 5. 2% Na-hypochlorite Pseudomonas aerug. 6. 2% Na-hypochlorite Bacillus subtilis 7. 0,9% NaCl Staphylococcus aureus 8. 0,9% NaCl Pseudomonas aerug. 9. 0,9% NaCl Bacillus subtilis 10. 0,9% NaCl –––––––––––––

G. Microbes digesting pectine Pectinase is a general term for enzymes that break down pectin, a polysaccharide substrate found in the cell walls of plants. One of the most studied and widely used commercial pectinases is polygalacturonase. It is useful because pectin is the jelly-like matrix that helps cement plant cells together and in which other cell wall components, such as cellulose fibrils, are embedded. Therefore pectinase enzymes are commonly used in processes involving the degradation of plant materials. Pectinases are actually a mixture of enzymes, which, along with others such as cellulase, are widely used in the fruit juice industry where they are used to extract, clarify and modify fruit juices. Pectins are large polysaccharide molecules, made up (mainly) of chains of several hundred galacturonic acid residues. Enzymes in this pectinase group include polygalacturonases, pectin methyl esterase and pectin lyases. These pectinase enzymes act in different ways on the pectins, which are found in the primary cell walls and in the middle lamella. Pectins are well known also for their ability to form gels. Pectinases are produced during the natural ripening process of some fruits, where, together with cellulases, they help to soften their cell walls. These enzymes are also secreted by plant pathogens such as the fungus Monilinia fructigena and the soft-rot bacterium Erwinia carotovora, as part of their strategy for penetrating the plant host cell walls. In fact, the products of such enzyme assaults (oligosaccharins) act as a signal which induces uninfected cells to defend themselves. Among potato-type plants, the Erwinia species cause significant harm during the cultivation and storage of the plant. Erwinia carotovora (phytopathogene bacterium) is a Gram negative, rod shape bacterium, primary agent e.g. of soft rot. Exercise: G/1. Action of pectinases during anaerobic digestion - “hemp-bath” Make a bunch of hemp (or straw) with cotton yarn. Fill a test tube with water, take one spoon of soil, then place the hemp into it. Boil it for 3-5 minutes. Use a test tube where the soil is taken to the tube after the boiling process as controll. After a week of incubation from the supernatant, spore staining is suggested to see anaerobic spore-forming bacteria (Clostridium spp.). Effect of boiling: 1. entering the water into plant fibers. 2. kill most non-spore-forming bacteria from soil.

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G/2. Soft-rot test After washing, cut slices of potato and carrot and put them into Petri dishes. Inoculate them with suspension of Erwinia carotovora (100 µl respectively). As control, pipette the same amount of distilled water to one of the slices. Incubate for a week at 28°C. Evaluate data on next practical with Gram staining procedure.

H. Checking the pH of the sour-cabbage I. Evaluation of the anaerobic cultivation techniques; morphological description of

Clostridium colonies, determination of sulphate reducing bacterial count

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PRACTICAL VI

A.Transfer of the unknown strain to fresh media: Strains: Own unindentified strain Culture medium: Nutrient agar slants Equipment, material: Inoculating loop THE MICROSCOPE

The optical microscope, often referred to as the "light microscope", is a type of microscope which uses visible light and a system of lenses to magnify images of small samples. Optical microscopes are the oldest and simplest of microscopes. There are non-optical microscopes, which require chemical or ion staining of non-living samples, and can magnify exponentially greater than the optical microscope (e.g. electron microscope).

The components of the microscope

All optical microscopes share the same basic components:

• The eyepiece - a cylinder containing two or more lenses to bring the image to focus for the eye. The eyepiece is inserted into the top end of the body tube. Eyepieces are interchangeable and many different eyepieces can be inserted with different degrees of magnification. Typical magnification values for eyepieces include 5x, 10x and 2x. In some high-performance microscopes, the optical configuration of the objective lens and eyepiece are matched to give the best possible optical performance. This occurs most commonly with apochromatic objectives.

1. ocular lens, or eyepiece 2. objective turret 3. objective lenses 4. rough adjustment knob 5. fine adjustment knob 6. object holder or stage 7. mirror 8. diaphragm 9. condenser

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• The objective lens - a cylinder containing one or more lenses, typically made of glass, to collect light from the sample. At the lower end of the microscope tube, one or more objective lenses are screwed into a circular nose piece which may be rotated to select the required objective lens. Typical magnification values of objective lenses are 4x, 5x, 10x, 20x, 40x, 50x and 100x.

• The stage - a platform below the objective which supports the specimen being viewed. There is a hole in the center of the stage through which light passes to illuminate the specimen. The stage usually has arms to hold the slides.

• The illumination source - below the stage, light is provided and controlled in a variety of ways. At its simplest, daylight is redirected with a mirror. Most microscopes, however, have their own controllable light source that is focused through an optical device called a condenser, with diaphragms and filters available to manage the quality and intensity of the light.

The complete optical assembly is attached to a rigid arm which in turn is attached to a robust U shaped foot to provide the necessary stability. The arm is usually able to pivot on its joint with the foot to allow the viewing angle to be adjusted. Mounted on the arm are controls for focusing, typically a large knurled wheel to adjust rough focus, together with a smaller knurled wheel to control fine focus. Some microscopes have objective lenses, called an oil immersion lens. To use this lens, a drop of immersion oil is placed on top of the cover slip, and the lens is very carefully lowered until the front objective element is immersed in the oil film. Such immersion lenses are designed so that the refractive index of the oil and of the cover slip are closely matched so that the light is transmitted from the specimen to the outer face of the objective lens with minimal refraction. An oil immersion lens usually has a magnification of 50 to 100×. The actual power or magnification of an optical microscope is the product of the powers of the ocular (eyepiece), usually about 10×, and the objective lens being used. Compound optical microscopes can produce a magnified image of a specimen up to 1000× and, at high magnifications, are used to study thin specimens as they have a very limited depth of field. How a microscope works The optical components of a modern microscope are very complex and for a microscope to work, the entire optical path has to be set up and controlled very accurately. Despite this, the basic optical principles of a microscope are quite simple. The objective lens is, at its simplest, a very high-power magnifying glass i.e. a lens with a very short focal length. This is brought very close to the examined specimen so that the light from the specimen comes to a focus about 160 mm inside the microscope tube. This creates an enlarged image of the subject. This image is inverted and can be seen by removing the eyepiece and placing a piece of tracing paper over the end of the tube. By carefully focusing a brightly lit specimen, a highly enlarged image can be seen. It is this real image that is viewed with the eyepiece lens that provides further enlargement. In most microscopes, the eyepiece is a compound lens, with one component lens near the front and one near the back of the eyepiece tube. This forms an air-separated couplet. In many designs, the virtual image comes to a focus between the two lenses of the eyepiece, the first lens bringing the real image to a focus and the second lens enabling the eye to focus on the virtual image. Limitations of light microscopes At high magnifications with transmitted light, point objects are seen as fuzzy discs surrounded by diffraction rings. These are called Airy disks. The limit of resolution (Resolving power of

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a microscope) is therefore taken as the ability to distinguish between two closely spaced Airy disks (or, in other words the ability of the microscope to reveal adjacent structural detail as distinct and separate). It is these impacts of diffraction that limit the ability to resolve fine details. The extent of and magnitude of the diffraction patterns are affected by both the wavelength of light (λ) and the refractive materials used to manufacture the objective lens and the numerical aperture (NA or AN) of the objective lens. There is therefore a finite limit beyond which it is impossible to resolve separate points in the objective field. Assuming that optical defects in the whole optical set-up are negligible, resolution is indicated with d. nxsinα = numerical aperture (NA) Usually, a λ of 550 nm is assumed, corresponding to green light. With air as medium, the highest practical NA is 0.95, and with oil, up to 1.5. In practice the lowest value of d obtainable is around 0.2 micrometres or 200 nanometers. BACTERIAL MORPHOLOGY The observation of the characteristics of cell morphology is of great importance in the classification of bacteria with traditional taxonomical methods. These microorganisms cannot be identified solely by morphological characteristics, since the bacterial cells can only be assigned to a limited number of categories (Table 6.1). Bacteria are µm-sized organisms, where cell size is an important aspect of a thorough morphological characterization. The size and shape of the cells are observed by staining. The culturing circumstances, the age of the culture, the physiological condition of the bacterial cells can each alter the size and shape of the cells. The shapes of stained bacteria can usually be identified as rods, cocci or spirals. An average rod shape bacterium is 2-5 µm long and 0,5-0,8 µm in diameter. The average diameter of a sphere-shaped bacterium is 0.8 µm.

d = 1.22λ / 2nxsinα

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COCCUS (SPHERE) Micrococcus Following cell division, cells separate (singles) Micrococcus luteus Diplococcus Following cell division, cells remain in pairs Neisseria gonorrhoeae Streptococcus Chain of cocci Streptococcus lactis Staphylococcus Grapelike cluster of cocci Staphylococcus aureus Tetragenus Cell division on 2 planes, cocci in tetrads Planococcus Sarcina Cell division on 3 planes, cocci in aggregates

(packets) of eight Sarcina lutea

ROD (BACILLUS) Shape and size are very variable

long-short, wide-thin, coccoid, irregular

Bacillus megaterium Pseudomonas sp. Haemophilus influensae Corynebacterium sp.

CURVED ROD (SPIRAL SHAPE) Vibrio Cell with quarter or half a turn Vibrio comma Spirillum Rigid cell wall, moving with flagella, cell with

one or more turns Spirillum volutans

Spirochaeta Flexible cell wall, inside flagella, cell with one or more turns

Treponema palidum

THREADLIKE Actinomyces have branching cells, forming

bacterial hyphae and their network (mycelium) Streptomyces sp. Nocardia sp.

VARIABLE Intermediate forms Rhodococcus sp.

Table 6.1. Morphology of bacteria

B. STAINING

Staining is commonly used to facilitate or observe specific organism or intracellular features. Staining is generally carried out on dead, fixed cells. Extra contrast between different features in a given specimen may be achieved with counter-staining. In negative staining, the background is stained or made opaque. In light microscopy most substances used for staining are themselves colored and they import their color to the specimen in various ways. Some staining procedures depend on in situ chemical reaction to produce color. Others involve the deposition of metallic silver (Spirochaeta spp. ) or of a dye - containing complex (flagella - stain ). The usefulness of a given stain may depend on its ability to stain some type of cell or cell component more effectively than others;, thus e.g. certain stains are commonly used for staining protein, nucleic acid, lipid and cellulose. Whether or not a given dye can obtain a particular cell or intracellular feature may depend not only on the nature and solubility of the dye; but on factors such as permeability or integrity of the cell membrane. Another important factor is pH, thus e.g. a protein pH below its isoelectric point can be stained by an acidic dye and at a pH above it’s isoelectric point by a basic dye. The differential staining effect of some composite stains depends on pH and on the admixture of component dyes in their correct proportion.

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B.1. SIMPLE STAINING Most commonly fixed and stained smears are used for the study of cell morphology, intracellular constituents and structures. The chemistry of staining is based on the principle that different charges attract each other, while similar charges repel. In an aqueous environment, with the pH at 7, the net electrical charge produced by most bacteria is negative. The dyes that are applied for staining could be acidic dyes, basic dyes and neutral dyes according to their chemical characteristics. Each dye contains a cation (positive charge) and an anion (negative charge) where either one could be the chromophore (the part of the molecule that is brightly colored). Within the bacterial cells, the dyes are bound mostly by the proteins and nucleic acids (around neutral pH carrying a negative charge). Since acidic dyes carry a negative charge on their chromophore, the bacterial cell (also negatively charged) rejects these dyes, so that they can only stain the background of the cells. These so-called negative dyes include: Eozin Y. Negative staining results from staining with the black colored India Ink and Negrozin because of the colloidal size of the dye particles which therefore cannot enter the cell. The chromophore of the basic dyes have a positive charge and result in an even staining of bacterial cells (positive dyes), since it binds to the cell’s proteins and nucleic acids. Basic dyes include safranin (red), methylene blue (blue), crystal violet (violet), malachite green (green). Steps in detail:

1. Remove grease from the slides 2. Sign the slide 3. Make smears 4. Fix the smear 5. Stain with basic dye 6. Wash with tap water 7. Dry 8. Microscopy

Exercise: staining own bacterial strain, Micrococcus luteus, Bacillus cereus, Escherichia coli

B.2. DIFFERENTIAL STAINING I. Gram-staining - an important bacteriological staining procedure discovered empirically in 1884 by Danish scientist Christian Gram. When bacteria are stained with certain basic dyes, the cells of some species (Gram - negative) can be easily decolorized with organic solvents such as ethanol or acetone, while cells of Gram-- positive species restrict decolorization. The ability of bacteria to either retain or lose the stain generally reflects fundamental differences in the cell wall and is an important taxonomic feature, Gram staining is therefore used as an initial step in the identification of bacteria. The integrity of cell wall is necessary for Gram-positivity, disrupted Gram - positive cells often do not retain the dye. The cells of some bacteria are strongly Gram-positive when young, but tend to become Gram-negative in ageing cultures (e.g. Bacillus cereus, Clostridium spp.) this may reflect degenerative changes in the cell wall. Some bacteria give a Gram-variable reaction, they are sometimes G-positive, sometimes G-negative; this could reflect e.g. minor variation in staining technique or rather changes in cell wall thickness, etc.

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Color changes due to the different steps of Gram staining Gram positive cell Gram negative cell Fixed smear - - Staining with crystal violet + + Treatment with Lugol + + Washing with ethanol + - Staining with safranine + - Steps in detail:

1. Remove grease from the slides 2. Sign the slide 3. Make smears 4. Fix the smear 5. Stain with crystal violet (1’) 6. Wash with tap water 7. Treatment with Lugol solution (KJ-J) 8. Wash with tap water 9. Decolorize with ethanol 10. Wash with tap water 11. Stain with safranine (1’) 12. Wash with tap water 13. Dry it 14. Microscopy

Exercise: staining own bacterial strain, Micrococcus luteus, Bacillus cereus, Escherichia coli C. BACTERIAL FLAGELLAE Movement of bacteria with flagellae is observed in liquid media, or in semisolid media while gliding motility is observed only on solid surfaces. THE ABILITY OF MOTILITY I. Indirect method: inoculate tubes of motility medium by stabbing the medium to a depth of about 5cm. Incubate at the optimum temperature and below. Motile organisms migrate through the medium which become turbid; growth of non-motile organisms is confined to the stab. Motility Test Medium Bacto Tryptose 10,0 gr. Sodium Chloride 5,0 gr. Bacto Agar 5,0 gr. Destilled Water 1000,0 ml pH = 7,2 Exercise: study own bacterial strain Fig. 6.1. Motility of bacteria studied by indirect method

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D. Checking the pH of the sour-cabbage – spreading to Rogosa agar medium ROGOSA AGAR is used for the isolation, enumeration and identification of Lactobacilli. This medium is selective, modified by Rogosa to contain high levels of Sodium acetate and Ammonium citrate at a low pH which inhibits most microorganisms, including Streptococci and molds and limits swarming but allows for the growth of Lactobacilli. Contains sacharose, arabinose and dextrose as fermentable carbohydrates, as carbon and energy sources; Tryptone provides nitrogen, vitamins, minerals and amino acids; Yeast extract provides vitamins, particularly B-group and other trace elements essential for growth; the Sulfate salts provide inorganic ions; Sorbitan monooleate is a surfactant; Monopotassium phosphate is the buffer. Bacteriological agar is the solidifying agent. Direct inoculation or plate count methodologies can be used. Inoculate medium and incubate at 35 ± 2°C for 24 – 48 hours. - Gram staining from supernatant of the cabbage

F. Movie demonstrating size, shape and motility, etc. of microbes

36

PRACTICAL VII

A. Transfer of the unidentified strain to fresh media (2 copies): Strains: Own unindentified strain Culture medium: Nutrient agar slants Equipment, material: Inoculating loop B. Differential staining II. Acid-fast staining or Ziehl-Neelsen staining - this staining can be used for detection of acid-fast bacteria e.g. Mycobacterium spp., Nocardia spp., etc., which is stained with an acid-fast stain, (mainly carbolfuchsin), cannot be decolorized by mineral acids or by mixtures of acid and ethanol. Purpose: The acid-fast stain is a differential stain which distinguishes organisms with waxy cell walls that can resist decolorization with acid alcohol. Acid-fast bacteria have a waxy substance called mycolic acid in their cell walls which makes them impermeable to many staining procedures, including the Gram stain. These bacteria are termed "acid-fast" because they are able to resist decolorization with acid alcohol. Carbol fuchsin contains phenol to help solubilize the cell wall. Heat is also applied during the primary staining to increase stain penetration. All cell types will take up the primary stain. The cells are then decolorized with acid-alcohol, which decolorizes all cells except the acid-fast ones. Methylene blue is then applied to counterstain any cells which have been decolorized. At the end of the staining process, acid-fast cells will be reddish-pink, and non-acid fast cells will be blue. (Note: Acid-fast stains are performed on smears that have been heat-fixed.) Steps in detail:

1. Make smear and fix it 2. Heat it with carbolfucsin for 10’ 3. Wash with water 4. Decolorize with acid alcohol 5. Wash with water 6. Counterstain with methylene blue 7. Wash with water 8. Dry 9. Microscopy

Staining: Own bacterial strain, Rhodococcus rhodocrous; Staphylococcus aureus

C. Structural staining – Schaeffer-Fulton spore staining

Bacterial endospores are highly resistant, thick walled structures formed by vegetative cells during a process called sporulation. They are highly resistant to radiation, chemical agents, extremely high temperatures, dessication, and other normally harmful environments. Several bacterial genera are capable of producing endospores; Bacillus and Clostridium are the two most common endospore-producing genera. Due to the highly resistant nature of endospores, it is necessary to steam stain into them. The most common endospore

Heat + Carbol Fuchsin

Acid alcohol

Methylene blue

Acid-Fast Non-AF

Fig. 7.1. Acid fast staining

37

staining technique is the Schaeffer-Fulton method. Once the endospore has absorbed the stain, it is resistant to decolorization, but the vegetative cell is easily decolorized with water (leaving the vegetative cells colorless). Finally, the vegetative cells are counterstained with safranin to aid in their visualization. When viewed under a microscope, the endospores appear green, while the vegetative cells are red or pink. The steps in the endospore staining technique are listed below.

1. Prepare smear, fix with heat 2. Steam with malachite green 3. Wash with water 4. Counterstain with safranin 5. Wash with water 6. Dry 7. Microscopy

Exercise: Bacillus cereus; Saccharomyces cerevisiae; own bacterium D. Examination of living bacteria [the ability of motility] Hanging - drop preparation: organisms suspended in a drop of water and placed in the center of a coverslip. A special slide with a hollow depression in the center is used. The entire slide with the coverslip is quickly turn over so that the drop of culture is in the center of the depression. Before observation fix the coverslip with Vaseline/paraffinedrops to the cavity slide. - when observing live bacteria, be careful not to confuse motility with Brownian movement resulting from bombardment with water molecules. In Brownian movement, the organisms all vibrate at about the same rate and maintain a fairly constant spatial relationship with one another, whereas bacteria that are definitely motile progress continuously in a given direction. - motility can be observed most satisfactorily in young cultures [24 or 48 hours] because older cultures tend to become non-motile . An old culture may become so crowded with inert living and dead bacteria that it is difficult to find a motile cell. In addition, the production of acid or other toxic products may result in the loss of bacterial motility in older cultures.

38

PRACTICAL VIII

A.Transfer of the unknown strain to fresh media: Strains: Own unindentified strain Culture medium: Nutrient agar slants Equipment, material: Inoculating loop STUDY OF BACTERIAL ENZYMES INTRODUCTION In this exercise microbial physiology, the biochemistry of cells are studied. Microorganisms, like all living things, may modify their environment to some extent and utilize chemicals in solution as source of energy and as building blocks for growth and reproduction. With the help of the biochemical test, we would like to determine which substances the microbe strains can utilize and what products they produce in the meantime. The chemical end-products of some of the enzymatic reactions may be measured, or in other cases the disappearance of certain substances from the medium can be detected. What reactions a species can perform is determined by its genetic makeup. The DNA carries a pattern for all the enzymes (biological catalysts) that the microorganisms can produce. Enzymes are substances that alter the rate of chemical reactions, generally making them happen faster. Enzymes located inside the cells are called endoenzymes or intracellular enzymes. Those excreted outside of the cells are exoenzymes or extracellular enzymes. By making a series of different tests, a pattern of activity can be established (which in turn reflects the enzymatic makeup of the microorganisms), and this fingerprint aids in the identification and differentiation of the microorganism from closely related species. B. EXAMINATION OF RESPIRATORY CHAIN ENZYMES Energy can be produced by the cell through oxidation which in biological systems is accomplished primarily by the removal of hydrogen and electrons. Hydrogen is removed from the substrate (the substance is oxidized) and transported via various respiratory enzymes to a final hydrogen acceptor. Hydrogen and electron transport supply energy for the cell. The transfer of hydrogen and the concentration of energy are carried out through a series of oxidation-reduction reactions. Biological oxidation occurs with oxygen or some others substances as the final hydrogen acceptor. Respiration is a biological oxidation occurring with atmospheric oxygen (aerobic respiration) or inorganic compounds such as nitrates or sulfates (anaerobic respiration). Fermentation is biological oxidation that occurs when the initial hydrogen donor (usually a carbohydrate) is broken down and one or more of the dissemination products serve as the final hydrogen acceptor. Whether a cell respires or ferments depends generally upon the availability of terminal electron acceptors and the presence of the enzymes necessary to reduce them. Obligate aerobes require atmospheric oxygen for the final hydrogen acceptor and will grow only in its presence.

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B/1 CATALASE ACTIVITY Catalase is an enzyme found in most bacteria. It catalyzes the breakdown of hydrogen peroxide to water and oxygen.

Hydrogen peroxide is toxic to cells. But it is also a common byproduct of metabolic reactions that take place in the presence of water and oxygen. Therefore most organisms that are able to survive in an atmosphere containing oxygen produce enzymes to degrade the peroxide. The major function of catalase within cells is to prevent the accumulation of toxic levels of hydrogen peroxide formed as a by-product of metabolic processes - primarily that of the electron transport pathway. Catalase negative organisms tend to be anaerobic. Important catalase–negative genera are Streptococcus, Leuconostoc, Lactobacillus, Clostridium and Mycoplasma.

Strains: Own unidentified strain (24h on agar slant), Proteus vulgaris, Micrococcus luteus, Bacillus cereus, Escherichia coli Materials: 3 % H2O2 solution

Exercise: Add a few drops of 3 % H2O2 onto 24h-old cultures. If the strain is catalase positive, the oxygen gas that has formed can be readily seen as white froth. B/2. CYTOCHROME OXIDASE An oxidase is an enzyme that reduces (adds electrons to) oxygen;. Cytochrome oxidase is the final link in the electron transfer system that provides adenosine triphosphate (ATP) during aerobic respiration. This enzyme receives electrons and passes them to oxygen, forming water. Many bacteria that live in the presence of oxygen produce cytochrome oxidase, but some do not. Among the Gram–negative rods, for example Pseudomonas, and bacteria closely related to it, are oxidase producers. Members of the family Enterobacteriaceae are not. In this exercise, a test strip impregnated with the oxidase reagent is smeared with the bacterial culture. In a positive test, oxidized cytochrome-e, formed as the result of cytochrome oxidase activity, oxidiezes p-aminomethylamiline to form a red pigmented product.

Strains: Own unidentified strain (24h on agar slant) Proteus vulgaris, Micrococcus luteus, Bacillus cereus, Escherichia coli Materials: Oxidase reagent, filter paper, Pt inoculating loop (or glass rod)

Exercise: Add a drop of oxidase reagent onto a filter paper. Let it impregnate the paper. Then smear some of the test microorganism onto the impregnated surface with the inoculating loop. (DO NOT use ordinary inoculating loops, since the Fe2+ ions can interfere with the test and result in a false positive reaction; use one made of Platinum or use a glass rod). If you observe purplish blue coloration within 30-60 sec., it is considered a positive reaction. Coloration that forms later than 1 min is considered negative.

40

B/3. METHYLENE BLUE REDUCTION Some bacteria are able to utilize methylene blue as a terminal electron acceptor when oxygen is not present. The removal of the oxygen from the broth and the formation of reducing substances during bacterial metabolism cause the color to disappear because methylene blue is reduced to the colorless leuco-methylene blue.

Strains: Own unidentified strain (24h on agar slant), Escherichia coli Materials: inoculating loop, methylene blue broth

Exercise: Inoculate a loopful of bacteria aseptically into methylene blue broth and incubate at 28°C for a week. The test is positive if the blue color starts to disappear from the bottom of the tube. If the broth is shaken, then the blue coloration will reappear since oxygen could dissolve back into the broth. C. DECOMPOSITION OF BIOPOLYMERS

C/1. CASEASE ACTIVITY (casein hydrolysis) Casein is the primary protein of milk. It exists as a colloidal suspension that gives milk its opaque cloudiness. The protein is too big to enter the cell, so uptake is only possible following extracellular hydrolysis. Many bacteria are equipped with enzymes that hydrolyze this protein into more soluble derivatives. The hydrolyzed protein, and subsequently the milk itself, become nearly transparent. Protein breakdown, sometimes called peptonization, is a useful reaction in the identification of bacteria.

Strains: Own unidentified strain (24h on agar slant), M. luteus, B. subtilis Materials: HCl containing HgCl2 solution, skim milk-agar plate

Exercise: Make a spot inoculation (making an X with the loop) on the surface of skim milk agar plate by placing a loopful of bacteria near one edge of the surface. After incubation,

Milk agar

Proteolytic zone No degradation

41

colonies of organisms that are able to digest casein (proteolytic microbes) will be surrounded by clear zones. Areas in which casein has not been degraded will remain slightly opaque. In some casesfalse positive reaction could result if the casein is just partly proteolyzed into para-casein, which also exhibits a clear zone. To eliminate such bad readings of the resultspour HCl containing HgCl2 solution onto the surface of the milk agar plate. This will denature proteins along with the para-casein so only areas where full proteolysis has occurred will remain clear. C/2. GELATINASE ACTIVITY The protein gelatin is a water soluble mixture of polypeptides that can be obtained from connective tissues and tendons of animals by boiling. The substance has a gel forming characteristic. Many bacteria are capable of producing gelatinase that hydrolyzes gelatin and breaks down gelatin to utilizable amino acids.

Strains: Own unidentified strain (24h on agar slant) Materials: HCl containing HgCl2 solution, gelatin-agar plate

Exercise: Make a spot inoculation (making an X with the loop) on the surface of gelatin agar plate by placing a loopful of bacteria near one edge of the surface. After incubation (a week) the agar plates are flooded with HCl containing HgCl2 solution. Within minutes the HgCl2 denatures proteins, forming an opaque precipitate with gelatin. Clear zones will appear where the gelatin has been broken down. C/3. α-AMYLASE ACTIVITY Starch is a polysaccharide that is abundant in nature; a rich source of carbon and energy. It is a common reserve carbohydrate supplying the nutritional need of plant roots and seeds. But the polysaccharide is too large to cross bacterial semipermeable cell membranes. Amylases cleaves large starch molecules into monosaccharide and disaccharide units small enough to enter into a bacterial cell. There the sugars are degraded by endoenzymes, releasing energy and carbon.

Strains: Own unidentified strain (24h on agar slant) Materials: Iodine solution, Starch agar plate

Exercise: Make a spot inoculation (making an X with the loop) on the surface of starch agar plate by placing a loopful of bacteria near one edge of the surface. After incubation (one week) the agar plates are flooded with iodine solution. Evaluation of the results is based on the reaction of starch with iodine which forms a blue coloration. Clear zones around the inoculated area indicate a positive reaction, where amylase has destroyed starch. C/4. LYPOLYTIC ACTIVITY (TWEEN 80 HYDROLYSIS) Lipids are found in every kind of cell membrane, and they are common in plant and animal nutrient storage compounds. The common lipids decomposed by microorganisms are triglycerids and phospholipids. The triglycerids are esters of glycerol and fatty acids. The reaction involved in their hydrolysis occurs as follows:

42

CH

CH

CH

2

2

O

O

O

C

C

CO

O

OR

R

R

'

''

3 H2O

CH2

OH

CH OH

CH2

OH

+

RCOOH

R' COOH

R'' COOH

triglyceride lipase glycerol 3 fat ty acids molecules (Tween 80 is the oleic acid ester of a polyoxyalkylene derivative of sorbitan) One of the main groups of esterases are the lipases. Most methods described for the disposal of bulk wastes employ various methods for the removal of fats and greases because their presence would markedly increase the requirement for available oxygen and in turn slow down the main process of sewage decomposition.

Strains: Own unidentified strain (24h on agar slant) Materials: Tween 80 agar plate

Exercise: Make a spot inoculation (making an X with the loop) on the surface of Tween 80 nutrient agar plate by placing a loopful of bacteria near one edge of the surface. After incubation evaluate the results on the basis of the presence or absence of Ca-oleate crystals near the area of inoculation. In the case of a positive reaction by the hydrolysis of Tween, oleic acid is freed and the calcium ions present in the media form Ca-oleate precipitation zones around the inoculation. C/5. DNASE ACTIVITY Organisms may be incubated on plates of DNA agar. After incubation flood the plates with 1M-HCl; a positive result shows as a clear zone around the growth the surrounding medium - and a negative result of the rest - is opaque.

Strains: Own unidentified strain (24h on agar slant) Materials: 1N HCl solution, DNA containing agar plate

Exercise: Make a spot inoculation (making an X with the loop) on the surface of DNA containing agar plate by placing a loopful of bacteria near one edge of the surface. After incubation pour 1N HCl solution on the surface of the plates. The acid denatures and precipitates DNA, so where the reaction is positive, and DNA has been broken down, there will be clear zones around the inoculated area. The test is negative if there is no clear zone visible around the spot inoculation. D. HAEMOLYSIS The majority of bacterial pathogens produce erythrocyte destroying (haemolysis) enzymes. There are two types of hemolysis: α–, and β–haemolysis. If the erythrocytes are completely destroyed, the bacterial colonies are surrounded by clear zones; this is the case for β

43

hemolysis. In other cases, there is a greenish–brownish zone visible around the inoculated area, which can be accounted for the partial degradation of red blood cells.

Strains: Escherichia coli, Staphylococcus aureus, Streptococcus faecalis, Streptococcus sp., Bacillus cereus, Bacillus cereus, Own unidentified strain Materials: Blood-agar plate, inoculating loop

E. CARBOHYDRATE (MONO AND OLIGOSACCHARIDES) DEGRADATION E/1. HUGH–LEIFSON TEST: GLUCOSE OXIDATIVE AND FERMENTATIVE DEGRADATION The oxidation–fermentation test is used to distinguish between fermentation – for which oxygen is not necessary – and respiration – for which oxygen is used as terminal electron acceptor. A semisolid agar medium together with a sugar glucose and bromthymol blue indicator that changes from blue to yellow when the environment becomes acidic. For each test, two agar deep tubes are needed: the aerobe tube and the fermentative tube, which is sealed with a thick layer of paraffin to prevent oxygen from penetrating into the medium.

Strains: Own unidentified strain Materials: Hugh-Leifson semisolid agar deep tube (one aerobe and one anaerobe

(sealed with paraffin), both having a bluish-green colour) per strain, inoculating loop

Exercise: Inoculate both the aerobe and the fermentative tubes with a loopful of the given bacteria by making a stab inoculation deep inside the agar tubes. When inoculating the fermentative agar deep, you must stab through the paraffin layer, so be careful because during the sterilization of the inoculating loop the adhering paraffin might catch fire and squirt. Incubate the tubes at 28°C for a week. Evaluation of the results is as follows: Fermentation: The fermentation of the glucose starts at the most anaerobic part of the tube (the bottom) so read the fermentative agar deep tube from the bottom. A yellow coloration at the bottom of the fermentative tube indicates a week positive reaction. Acid is formed in both tubes for fermentation. Oxidation: Aerobic utilization of glucose starts at the most aerobic part of the media (top of the tube), therefore starting the readings of the oxidative tube from the top. A yellow color in the upper portion of the tube indicates aerobic utilization of glucose. E/2. METHYL RED AND VOGES–PROSKAUER TESTS This test is preformed in MR–VP Medium containing glucose, peptone, phosphate buffer and methyl red indicator. This MR–VP test is used to identify organisms able to produce acetoin from the degradation of glucose during a 2,3-butanediol fermentation. Both the MR and VP tests are especially useful in differentiating between members of the Enterobacteriaceae.

KOH O2

Acetoin + α-napthol →→→ diacetyl-creatin complex (red colour)

Strains: Own unidentified strain Materials: MR–VP Medium, α–napthol (Barritt reagent A), 40% KOH (Barritt

reagent B), empty test tube, methyl red indicator

44

Exercise: Inoculate a loopful of the test microbe into MR-VP medium. Incubate the tubes at 28°C for a week. When evaluating the results, take 1 ml of sample into a new test tube. After 48 hours of incubation, Barritt's Reagent A (α-napthol) and Barritt's Reagent B (potassium hydroxide) are added to the sample. After shaking the tube gently for aeration, formation of a red color after approximately 20 min. will indicate a positive VP reaction. Add 3-4 drops of methyl red indicator solution to the remaining MR-VP medium. A lasting red coloration is considered to be positive MR reaction. E/3. AESCULIN HYDROLYSIS Esculin (β-glucose-6,7-dihydroxycoumarin) hydrolysis is a useful test in the differentiation of both gram-positive and gram-negative bacteria. Hydrolysis is indicated by the production of a brownish-black colored compound, due to the combination of ferric ions (Fe3+) with the hydrolysis product esculetin (6,7-dihydroxycoumarin) as indicator.

hydrolysis β-glucose-6,7-dihydroxycoumarin →→ glucose + 6,7-dihydroxycoumarin

(Esculetin) Strains: Own unidentified strain Materials: Esculin broth, inoculating loop

Exercise: Inoculate a loopful of the test microbe into Esculin broth. Incubate the tubes at 28°C for a week. A blackish, dark brown colloidal precipitate forms in the test tube if the test is positive. If there is no precipitate, the test is negative. F. AMINO ACIDS F/1. H2S PRODUCTION (CYSTEIN DESULFHYDRASE) Bacteria that produce the enzyme cystein desulfhydrase are able to strip the amino acid cystein of both its sulfhydryl (-SH) and amino groups. The reaction yields hydrogen sulfide (H2S), ammonia ( NH3 ) and pyruvic acid. Hydrogen sulfide reacts with heavy metals such as lead or iron to form a visible, black precipitate. HS⎯ CH2 ⎯ CHNH2⎯ COOH → H2S ↑+NH3+CH3⎯ CO⎯ COOH Cystein gas pyruvic acid H2S + Pb(CH3COO)2 → PbS + 2CH3COOH

black precipitate

Strains: Own unidentified strain Materials: Nutrient broth, inoculating loop, filter paper impregnated with

Pb(CH3COO)2

Exercise: Inoculate a loopful of the test microbe into cystein containing broth. Place the test stripe (lead acetate impregnated filter paper) into the tube, so that it does not hang into the liquid medium and it is fixed between the cap or plug and the side of the test tube. (Use gloves when handling the filter paper). Incubate the tubes at 28°C for a week and examine the filter paper. The test is positive if the paper turns black, the test is negative if there is no coloration of the filter paper.

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F/2. INDOLE PRODUCTION

Some bacteria hydrolyze tryptophan to pyruvic acid, which is then metabolized in a pathway similar to the familiar Krebs pathway. A byproduct of that hydrolysis is indole, which is excreted by the organism. Pure tryptophan is not ordinarily used in the test medium. Instead, tryptone - a digestion product of certain proteins - is used as the substrate since it contains considerable amount of tryptophan. The presence or absence of the responsible enzyme, tryptophanase, is important in the differentiation of enteric bacteria such as Eschericia coli.

CH2

CH

NH2

COOH

+ H2

ONH*

*NH

+ CH3

CO COOH + NH3

pyruvic acidindole

tryptophan

+ H2

O

Strains: Own unidentified strain Materials: Nutrient broth, inoculating loop, Kovács reagent

Exercise: Inoculate peptone broth with the test microbe and incubate the tubes at 28°C for a week. Add 0,2 ml of Kovács reagent (5% para-dimethyl-amino-benzaldehyde in 75% amyl alcohol, 25% concentrated hydrochloric acid) to the test tube, mix well and let it rest for about 5 min. A positive test is indicated by a red coloration in the upper alcohol layer. The test is negative if the upper alcohol layer is yellow.

G. PHOSPHATASE ACTIVITY This test was used by Barben & Kirby to aid the identification of pathogenic staphylococci ; they found a high degree of correlation between phosphatase and coagulase production.

phosphatase Na- phenolphthalein-phosphate → phosphate + phenolphthalein

(red in alkaline solution)

Strains: Own unidentified strain Materials: Na- phenolphthalein-phosphate agar plate, inoculating loop, ammonia

Exercise: Inoculate a loopful of the test microbe onto the surface of Na- phenolphthalein-phosphate agar plate and incubate the plates at 28°C for a week. Turn the agar plates upside-down and pipette a drop of ammonia onto the top of the Petri-dish. If the test bacteria is phosphatase positive, free phenolphthalein liberated by phosphatase reaction reacts with the ammonia and phosphatase positive colonies become bright pink.If the test is negative, there is no visible color change.

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H. Cultivation of antibiotic producer microbe: Starting the shaken cultures of Streptomyces strains

47

PRACTICAL IX.

A.Transfer of the unknown strain to fresh media: Strains: Own unindentified strain Culture medium: Nutrient agar slants Equipment, material: Inoculating loop

B. Determination of the lysosime content of an egg 

Lysosime is an enzyme responsible for the digestion of murein of bacteria, produced by several organisms (plants, animal tissues, bacteriophages, bacteria etc). Test will be evaluated by the decrease of the optical density of Micrococcus luteus suspension.

1. Making suspension from M. luteus (24-hour culture) in 5 ml phosphate buffer. 2. Egg white is diluted to 1000x with phosphate buffer. 3. Standard: lysosime solution (4µg/ml) 4. Making a dilution from samples as follows:

Half test tube 1 2 3 4 5 6 7

Buffer (ml) 0 2 3 3,5 3,75 3,88 3,94

sample (ml)a 4 2 1 0,5 0,25 0,12 0,06

M. luteus suspension (ml) 0,3 0,3 0,3 0,3 0,3 0,3 0,3

Gg white OD (30 min.)

Standard OD (30 min.) a egg white or lysosime solution

5. Pipette 0.3 ml M. luteus suspension to the dilution steps in 1minute intervalls and mix it.

6. Incubate at room temperature for 30 minutes. 7. After incubation, determine the optical density of solutions at 520 nm wavelength.

C. Antimicrobial gs These are chemicals used to treat diseases; they include antibiotics, a group of compounds originally produced by metabolic reactions of bacteria and fungi that prevent the multiplication of other microbes. Most antibiotics are produced by bacteria (genera Streptomyces and Bacillus and fungal genus Penicillium). Some of those are chemotherapeutic agens - may be defined as chemicals that, at concentrations toleraded by the host, can interfire directly with the proliferation of pathogenic micro-organisms. Thus the essential feature is one of selective toxicity. Some drugs are bacteriostatic, the inhibition of growth being reserved when the drug is removed, others are bactericidal, exerting an irreversible lethal effect. The distinction between bactericidal and bacteriostatic action is however not absolute, since some normally bactericidal agents appear to be bacteriostatic at relatively low concentrations.

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Antibiotics: were originally defined by Waksman (1945) as “chemical substances“ produced by microorganisms which possess the ability to kill or to inhibit the growth of bacteria and other microoganisms. Most of these compounds act by inhibiting the formation of a particular type of macromolecule in the microbial cell. For example, penicillus and cefalosporius inhibit peptidoglycan synthesis; streptomycin, chloramphenicol and the tertacyclines interfere with protein biosynthesis, refampicin and actinomycin D prevent nucleic acid synthesis, polymyxin B, valinomycin and gramicidin A inhibit the cell membrane function.

C/1.The use of Resistest discs ‐ Kirby‐Bauer Technique:  In this method a culture of known concentration is spreaded on an appropriate medium (Mueller-Hinton medium is widely used because it is an appropriate growth medium for most bacterial pathogens and it contains very few substances that inactivate antibiotics). Filter paper discs containing predetermined concentrations of an antimicrobial are placed on the seeded agar plate, with equal spacing between discs (Generally 4 discs / Petri dish). During incubation, the agent diffuses out of the disc, creating a concentration gradient that decreases according to distance away from the disc. After the incubation the organisms, sensitivity is measured on the basis of the size of the zone of inhibition (no growth) arround each disc. This measure is compared to values on a standard, and it indicates whetwer the microorganism is resistant, intermediate or sensitive to the agar. Sensitive means that the organism is inhibited by clinically attained concentrations of the antimicrobial; resistant means that the organisms is not inhibited; intermediate meant that special considerations are to be followed if the antibiotic is to be used.

R : resistant zone of inhibition with a diameter equal to or less than inner circle. I : intermediate zone of inhibition with a diameter greater than R. S : sensitive zone of inhibition with a diameter greater than outer circle .

I

49

Fig. 9.1 Determination of sensitivity to antibiotics

The Kirby-Bauer method: i./ standardized the concentration of antibiotics to be tested ; ii./ standardized the incubation time ; iii./ standardized the nature of inoculum iv./ standardized measurement of the inhibition zone. Antibacterials 1./ Alexander Fleming discovered penicillin in 1929, however, it was not tested in a human until 1941, when Florly and Clain purified enough penicillin to carry out clinical trials. Penicillin G became widely available after War World II. The development of the semisynthetic penicillins had to wait until the microbial production of 6-aminopenicillinic acid in the late 1950’s. 2./ The range of the microbes affected by an antimicrobial agent is its spectrum, antibiotics are either broad spectrum or narrow spectrum antimicrobials. 3./ Antibiotics inhibit the growth of (bacteriostatic) or kill (bactericidal) microorganisms by interfering with protein synthesis, nucleic acid synthesis, cell-wall synthesis, or cell-membrane function. 4./ Bacteria acquire resistance to antibiotics when a structure is altered so as to prevent antibiotic binding or the cell acquires genes (plasmid) encoding enzymes that inactivate the antibiotic. C/2. In vitro synergy between combinations of antimicrobial agents The addition of two or more antimicrobial agents to a microbial population sensitive to each of the individual compounds may have various outcomes. The overall inhibitory effect may be insignificantly different from that of the sum of the individual agents (indifference), it may show enhancement over that of the individual agents (synergy), or it may be considerably lower than that anticipated from the individual responses (antagonism). For example, combinations Trimethoprim and sulfonamides are murkedly synergistic to a variety of microorganisms, while polymyxin and sulphonamides show synergy with some Proteus species. Antagonism between nalidixic acid and nitrofurantoin has been reported for some Proteus isolates and Staphylococous aureus sensitivity to lincomycin is antagonized by erythromycin.

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The technique used in this experiment involves placing sensitivity disc soaked in solutions of different antimicrobial agents onto seeded agar plates such that they overlap, and examining the nature of any areas of growth inhibition surrounding the intersection after incubation. C. Antimicrobial compounds of plants (chamomilla, horse radish, onion, etc.) Many plants contain chemical compounds that inhibit the growth of microorganisms and this may play an important part in their resistance to pathogens. Different plants produce different compounds which vary in their antimicrobial action and microorganisms differ in their sensitivity to the compounds. Garlic is a good example of one of the many plants that contain such antimicrobial compounds. The technique used in this case involves placing slices of garlic onto the seeded agar plates and examining the nature of any areas of growth inhibition surrounding the garlic slice after the incubation. Exercise:

1. Spread Petri dish media with the adequate bacterial suspension 2. Place onion/garlic to it 3. OR: make a hole on the spreaded Petri dish and fill it with the adequate plant extract 4. Incubation

D. Detection of antagonism in cross streak experiments 1. antimicrobial producer strain 2. test organisms

E. Screening the filtrate of Streptomyces strain Microorganisms produce several primary and secondary metabolites. Some of them have antimicrobial activity. Testorganism: Staphylococcus aureus

1. Shake a given Streptomyces strain for 1 week, then filtrate it sterile. 2. OD of test organism must be 0.3 at 660 nm. 3. Add 1 ml S. aureus suspension to 100 ml melted (50°C) agar media, pour it to Petri

dishes and solidify it. 4. Make a hole in the infected plates, pipette to the holes the filtrate of Streptomyces. 5. Inhibition zones can be measured after 24 hours.

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F. Effect of heavy metals to bacteria Heavy metals (Ag, Cu, Ni, etc) are toxic for microorganisms already in low concentrations (denaturation of proteins). In the present exercise, ions of the given heavy metal will diffuse into the medium and thus produce an inhibition zone. Exercise:

1. Spread Petri dish media with the adequate bacterial suspension 2. Place a small piece of Cu plate on it 3. OR make hole on the spreaded Petri plate and fill it with the adequate solution

(CuSO4) Strains: Escherichia coli Staphylococcus aureus

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PRACTICAL X

Coliforms are good indicator organisms. They are not generally pathogenic, their presence shows that faecal contamination of water has occurred. Coliforms in the hygienic practice are defined as those facultative anaerobic, Gram-negative, non–endospore-forming, rod-shaped bacteria that ferment lactose, produce acid and gas within 48 hours at 37°C. Coliforms are members of the family Enterobacteriaceae (e.g. E. coli, Enterobacter aerogenes, Klebsiella pneumoniae). The values of coli-count and the coli-titer are used for the quantitative characterization of coliform organisms in a sample. Coli-titer is the smallest amount of water, from which coliform organisms can still be cultivated. Coli-count (Coli-index) is the number of coliform bacteria that can be cultivated from 100 ml of water sample. Streptococcus faecalis dies in water quickly, however its evaluation is important, since, as opposed to E. coli (which can be found almost anywhere), S. faecalis does not reproduce in the nonfecal environment. Thus its presence underlines the results of E. coli testing and indicates the fresh fecal contamination of water. Anaerobic spore forming Clostridium welchii stays viable in water for a long period of time, thus its presence in water indicates heavy and long-lasting fecalcontamination of water. A. THE TRANSFER OF THE UNIDENTIFIED STRAIN TO FRESH MEDIA (2 COPIES): Strains: Own unindentified strain Culture medium: Nutrient agar slants Equipment, material: Inoculating loop B. MICROBIOLOGICAL EXAMINATION OF SURFACE WATER; HYGIENIC CONTROL B/1. COLI-COUNT DETERMINATION BY MEMBRANE FILTER TECHNIQUE A membrane filter with a 0.45µm pore-size is generally used to remove bacteria from solutions.

Exercise: Filter 100 ml of a water sample through a 150µm thick and 0.45µm pore-sized sterile membrane filter. Remove the membrane filter and place it facing up onto the surface of

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differential media (endo- or eosin-methylene blue agar plates). Incubate the plates at 37°C for 24 hours and count the number of colonies formed. Knowing the volume of water that has been filtered, we can estimate the Coli-count of 100 ml water sample.

Sample: water sample (river water, well water) Materials: graduated cylinder (to measure a 100 ml of water), 0,45µm pore-size

membrane filter (for filtering tap water or natural water samples), sterile filtering unit, Endo agar Petri plates (LES Endo Agar) or Eosin-Methylene Blue agar plates (EMB Agar), metal forceps (use alcohol for flaming) B/2. COLI-COUNT DETERMINATION BY MPN TECHNIQUE USING LMX-BROTH IN 96 WELL MICROTITER PLATES The borderline dilution method is based on a serial dilution prepared form a sample and inoculation of the special (giving a typical reaction for the given group of microorganisms) differential media with the same amount of suspension of each dilution. Following the predefined incubation conditions (time and temperature), the most probable bacterial count (MPN most probable number) could be estimated from the number of tubes indicating bacterial growth by mathematical/statistical calculations. This method presumes that the last dilution, the last test tube where bacterial growth could be observed, contains only one cell. The probable bacterial count of 1 ml of sample with this technique can be estimated. During this practical, we employ LMX medium containing MUG substrate which can be cleaved by the GUD enzyme, cleaving off a compound that gives fluorescence under 366 nm wavelength UV light. Among the Gram-negative bacteria, 96% of E. coli, 100% of entorotoxic E. coli, 44% of certain Shigella species and 17% of bacteria belonging to the Salmonella genus produce the enzyme

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Sample: water sample (river water, well water) Materials: LMX broth, 50 ml Erlenmeyer flask, pipette and tips, UV-lamp of 366

nm wavelength, 96 well microtiter plates, test tubes, vortex, circular shaker

Exercise: Prepare a serial dilution from the water sample as follows: Measure 3g/ml water sample into 27 ml LMX broth in 50 ml Erlenmeyer flask. Homogenize the sample at 400 RPM (rounds per minute), at 20°C for 15 minutes on a circular shaker. Prepare an 8-member dilution series from the obtained suspension so that you carry 0,3 ml of the sample suspension into a test-tube containing 2,7 ml of LMX broth. Mix vigorously (vortex) then again transfer 0,3 ml suspension into a new test tube, and so on. From each dilution series (including the original sample), pipette 300 µl onto a 96-well microtiter plate in five parallels (5 test-tube MPN method). Incubate the microtiter plates at 37°C for 24 hours. Evaluation of the results: the E. coli count is determined by counting the fluorescent wells (under UV-lamp of 366 nm wavelength) of the plate and transforming it to the appropriate McCardy characteristic number.

Table 10. 1. McCardy-table B/3. EXAMINATION OF COLIFORMS ON ENDO- OR EOSIN-METHYLENE BLUE AGAR PLATES Eosin-Methylene Blue agar is one of the most efficient differential media. It differentiates lactose fermenting bacteria based on the fact that the acid produced by lactose fermentation precipitates eosin, which is colored by the methylene blue dye content of the medium, so that the bacterial colonies on the surface of the Eosin medium display a purplish blue coloration. The medium also contains sucrose which helps the groth of other microbes but later the dye inhibits growth. Within the Endo-agar: the dark red alkaline fuchsin is decolorized by sodium sulfite (Na2SO3). Acetaldehyde is one of the intermedier compounds during lactose fermentation,

55

which binds sulfite, thus the color of the fuchsin becomes visible. The typical coliform organisms dye the Endo-agar red and grow on the surface of the agar as dark red colonies having a metallic shine.

Materials: Endo agar Petri plates (LES Endo Agar) and Eosin-Methylene Blue agar plates (EMB Agar), 9 ml sterile water containing test tubes (for the preparation of dilution series), glass rod (alcohol for flaming), sterile pipet and tips

Exercise B/3: Prepare a serial dilution from the water sample and spread 0,1-0,1 ml of each dilution onto the surface of Endo-agar and Eosin-Methylene Blue agar plates. Incubate the plates at 37°C for 24 hours and evaluate the number of typical coliform colonies. B/4. CULTURING PSEUDOMONADS ON BROLACINE AGAR Member of the Pseudomonas family are widely distributed in soil and water. The Pseudomonads are Gram-negative, strictly aerobic, mostly obligate aerobic bacteria having polar flagella. Among them, Pseudomonas aeruginosa is a common bacterium of different waters, wastewater and contaminated water. For the demonstration of the members of the genus Pseudomonas, brolacin nutrient medium is used, which contains lactose, peptone, cystine and brom thymol blue indicator (green at neutral pH). The colonies of Pseudomonads appear brown in the centre and dye the surrounding area of the agar blue. The presence of Pseudomonads does not indicate fresh contamination, however it can often be found inside wet hospital units (taps, pipelines,etc.) and equipment. This microbe usually attacks patients who are weak, immune suppressive and received radiation or antibiotic treatment.

Materials: Brolacin agar, glass rod (alcohol for flaming), sterile pipet and tips Exercise: Spread 0,1-0,1 ml of each dilution onto the surface of Brolacin agar plates. Incubate at 37°C for 24 hours, observe the brown centered colonies with blue zones around them. Calculate a Pseudomonas-count from the amount of such colonies. B/5.DEMONSTRATION OF STREPTOCOCCUS OF FECAL ORIGIN USING SZITA-E-67 MEDIUM Members of the Gram-positive coccus the genus Streptococcus produce shorter-longer chains. Some of them are human and animal pathogens but healthy human or animal organisms can also carry these bacteria. In clinical, epidemiological laboratory practice, the Szita E-67 agar medium is generally used for the cultivation of Streptococcus. The selectivity of the Szita E-67 medium for Streptococcus is assured by the medium’s tellurite and Na-taurocolate content. Streptococcus faecalis reduces the tellurite within the medium and forms black colonies on the surface of the Szita E-67 agar, while other bacteria hardly growth at all.

Materials: Szita-E-67 agar, glass rod (alcohol for flaming), sterile pipette and tips Exercise: Spread 0,1-0,1 ml of each dilution onto the surface of Szita-E-67 agar plates. Incubate at 37°C for 24 hours, observe the characteristic black Streptococcus colonies. Calculate a Streptococcus-count from the amount of such colonies.

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B/6. DETERMINATION OF COLIPHAGES FROM SURFACE WATERS BY THE POUR-PLATE-TECHNIQUE Bacteriophage (viruses that infect bacteria), often called phage, are by far the most convenient viruses for laboratory investigations. Phages are rapidly replicated in their bacterial hosts. The phage that infects Escherichia coli is called coliphage. Coliphage is added to the bacteria in soft agar prior to pouring the plate, iff the coliphage lyse (burst) cells while Escherichia coli is multiplying in the overlay, plaques (clear areas) develop in the lawn of bacteria. The culturing of the bacteriophage of the fecal contamination indicating E. coli from an environmental sample is a new technique in the microbiological analysis of water samples. Exercise: Measure 100 ml water sample into a sterile flask and place it into a water bath of 45°C-47°C. Place 4 ml sterile CaCl2 solution into the same water bath for 10 minutes. Meanwhile, dissolve 0,05g of TTC (triphenyl-tetrazolium-chloride) in 5 ml 96% ethanol. Then measure 1 ml of the 1% TTC solution and 1,2 ml of the CaCl2 solution into 100 ml liquefied nutrient agar. Inoculate the water sample with 5 ml of a 24 h Escherichia coli (ATTC 13706) suspension previously shaken at 37°C. Carfully and slowly pour the agar into the water sample and slowly mix it, then pour it out into a big Petri dish avoiding the formation of any bubbles. The viable bacterial cells reduce TTC, which turns the agar red, while white plaques form where the phage has destroyed the bacterial cells. C. TSI MULTITEST MEDIUM FOR THE DIFFERENTIATION OF GRAM NEGATIVE BACTERIA Because of their complexity, multitest media can be used for testing several enzymatic activities simultaneously. TSI (Triple Sugar Iron) agar is generally used for the characterization of enterobacteria. The medium is rich in protein and contains 0,1% glucose, 1,0% sucrose and lactose, ferrous sulfate and phenol red indicator. Phenol red changes to yellow when the environment is acidic. The intestinal pathogens Salmonella and Shigella ferment dextrose but not sucrose or lactose. The most common Gram negative, non-pathogenic fecal rods do not share these characteristics.

Strains: Enterobacteria Materials: TSI agar slants, inoculating loop

Exercise: Inoculate the TSI agar slants with a stab inoculation as well as a zigzag strak inoculation on the surface of the agar slant. Place the tubes into the 28°C thermostat for 2-7 days. Evaluate the observed features of the agar as follows: Evaluation:

1. Yellow stab inoculation and red slant → acid production from glucose degradation (because only the fermentation of a small amount of glucose (0.1%) present in the medium happens, only the bottom, anaerobic zone becomes acidic.)

2. Yellow stab inoculation and yellow slant → acid production from lactose and/or sucrose degradation (because the fermentation of large amounts of sucrose and/or lactose (1%) happens, the whole nutrient medium becomes acidic and yellow.)

3. Formation of bubbles or cracks → intensive fermentation, gas formation 4. Red stab inoculation and red slant → nor carbohydrate degradation or gas formation 5. Black coloration → indicates H2S production

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TSI multitest media Stab

inoculation Slant surface inoculation

H2S production Gas production

Enterobacter yellow yellow – – Escherichia yellow yellow – ± Klebsiella yellow yellow – –

Proteus vulgaris yellow Yellow or red + – Serratia yellow Red or yellow – +/– Shigella yellow red – –

Salmonella typhi yellow red + –

Typical TSI reaction observed with some enterobacteria

D. EVALUATION OF THE EFFECT OF ANTIMICROBIAL AGENTS

58

PRACTICAL XI

A. THE MAIN PROCESSES INVOLVED IN THE NITROGEN CYCLE ARE:

Organic-N

NH3 N2 NO2- NO3

-

Assimilative nitrate reduction

Dissimilative nitrate reduction

Nitrification Nitrification

N-fixationAmmonificationandNH

assimilation3

The oxidation number of Nitrogen

-3 0 +1 +3 +5

N O2

FIG. 11.1. The nitrogen cycle

A/1. NITROGEN FIXATION: THE EXAMINATION OF NITROGEN FIXING MICROORGANISMS Biological nitrogen fixation is carried out by prokaryotic organisms. During the process of nitrogen fixation, the oxidation state of nitrogen atoms is reduced from 0 (nitrogen gas) to -3 (ammonia). The ammonia gained by nitrogen fixation could later be assimilated into organic compounds. The structure of the nitrogenase enzyme-system of the various nitrogen-fixing organisms differ only slightly, thus these organisms fix nitrogen practically through similar reaction. However, these nitrogen-fixing organisms can be divided into two major groups: free-living organisms (e.g. Azotobacter, Clostridium, Anabena) and symbionts (close association). The latter group consists mostly of the members of the genus Rhizobium, which is associated with leguminous plants (e.g. peas, beans, soybean). Free-living rods infect the root hairs of leguminous plants. The plant responds by producing nodules to wall off the infection. Inside these swellings, Rhizobium cells grow, becoming pleomorphic symbionts called bacteroids. Bacteroids have characteristic morphology and they fix atmospheric nitrogen. Among the nitrogen-fixing prokaryotes, aerobic, microaerophile and anaerobic microorganisms can be identified. Since the nitrogenase enzyme is very sensitive to the presence of oxygen, thus in an aerobic/oxic environment the intracellular oxygen tension should be reduced. The nodules contain leg-hemoglobin, which is responsible for the suitably low oxygen tension needed for the bacteroids. Cyanobacteria - morphology

Cyanobacteria, also known as blue-green algae, blue-green bacteria, is a phylum of bacteria that obtain their energy through photosynthesis. The name "cyanobacteria" comes from the color of the bacteria. They are a significant component of the marine nitrogen cycle and an important primary producer in many areas of the surface waters Cyanobacteria were

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traditionally classified by morphology into five sections, referred to by the numerals I-V. The first three - Chroococcales, Pleurocapsales, and Oscillatoriales - are not supported by phylogenetic studies. However, the latter two - Nostocales and Stigonematales - are monophyletic, and make up the heterocystous cyanobacteria. The members of Chroococales are unicellular and usually aggregate in colonies. The classic taxonomic criterion has been the cell morphology and the plane of cell division. In Pleurocapsales, the cells have the ability to form internal spores (baeocytes). The rest of the sections include filamentous species. In Oscillatoriales, the cells are uniseriately arranged and do not form specialized cells (akinetes and heterocysts). In Nostocales and Stigonematales, the cells have the ability to develop heterocysts in certain conditions. Stigonematales, unlike Nostocales include species with truly branched trichome.

Exercise: Study of water samples for Cyanobacteria; study of heterocysts

Heterocyst: Heterocysts are specialized nitrogen-fixing cells formed by some filamentous cyanobacteria, such as Nostoc punctiforme, Cylindrospermum stagnale and Anabaena sperica, during nitrogen starvation. They fix nitrogen from dinitrogen (N2) in the air using the nitrogenase enzyme in order to provide the cells in the filament with nitrogen for biosynthesis. Nitrogenase is inactivated by oxygen, so the heterocyst must create a microanaerobic environment. The heterocysts' unique structure and physiology requires a global change in gene expression. For example, heterocysts:

Examination of cyanobacterial morphology

Fig. 11.2. Morphology of cyanobacetria

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A.2. Examination of bacteroid morphology

Sample: root sample (leguminous plant) Materials: microscope slide, methylene-blue dye, scalpel and forceps

Exercise: Cut a nodule off the washed leguminous root sample using forceps and a scalpel. The active nodules have a slightly pink color, since they contain leg-hemoglobin. Gently press the nodules between two microscope slides. Air dry and heat fix the smear, then stain the smear with methylene-blue. Observe the large, irregular rods of the bacteroids under the microscope. B. DEMONSTRATION OF AMMONIFICATION During ammonification, some nitrogenous compounds (amino-acids, carbamide etc.) are transformed into ammonium ions and ammonia is freed. For such deamination reactions, bacteria utilize many enzymes with different substrate specificity (one way of getting rid of excess organic nitrogen). The remaining part of the molecule can be used for energy generation . Within the nitrogen cycle, ammonification is considered as a mineralization step from which ammonia and ammonium ion can be taken up and used for amino-acid synthesis by other organisms (plants, microbes) or it can be absorbed in the soil by humus-colloids.

NH3 + Potassium-mercury(II)-iodide → Mercury(II) [amido-triiodide-mercurate(II)]

orange complex Sample: own unidentified strain Materials: peptone broth, urea broth, sterile water in test tubes, Nessler-reagent

Exercise: Inoculate the peptone broth containing test tubes with 0,1-0,1 ml bacterial suspension and incubate the tubes at 28°C for a week. The presence of accumulated ammonia in the peptone broth can be demonstrated by adding a few drops of Nessler-reagent (an alkaline solution of Potassium-mercury(II) iodide). Weak positive reaction yields a yellow color, strong positive reaction results in a yellowish brown color and precipitation. C. DEMONSTRATION OF NITRIFICATION AND INHIBITION OF NITRIFICATION WITH ATU (ALLYL-THYOUREA)

During aerobic conditions, ammonia does not accumulate in soil or water. Beside the assimilation of ammonia (for amino-acid synthesis), certain bacteria can gain energy by utilizing ammonia as electron donor (as well as producing reducing potencial) in dissimilative processes. Nitrification is carried out by chemolitho-autotrophic bacteria (e.g. Nitrosomonas: NH3→NO2

- and Nitrobacter NO2-→NO3

-) and different heterotrophic microorganisms. Nitrite can be taken up by plants more easily than ammonia; however, because of its bigger mobility, nitrite can be leached from soil, deteriorating the quality of both surface and underground waters. Nitrification is a two-step process both chemically and biologically:

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Name of process Reaction Typical bacterial genus

Ammonia oxidation NH4+ + 1,5 O2 → NO2

- + H2O + 2 H+ Nitrosomonas, Nitrosospira

Nitrite oxidation NO2- + 0,5 O2 → NO3

- Nitrobacter, Nitrospira

The key enzyme in ammonia-oxidation is ammonia mono-oxigenase (Amo). The enzyme is active in the presence of copper ions. Allyl-thiourea (ATU) is a selective copper-chelator compound, thus ATU can inhibit the ammonia mono-oxigenase enzyme.

Sample: soil suspension, own unidentified strain Materials: Ammonia and nitrite containing broth, Nessler-reagent, Griess-Ilosvay-reagent

(nitrite reagent), zinc powder, ATU Exercise: Add ATU to half of the nutrient media, so that the final concentration would be 5 mg/l. Inoculate the media with 100 ml soil suspension. Incubate the samples at 28°C for a week. Evaluation of results: Transfer 1-1 ml of each broth to a new, empty test tube. Add some drops of Griess-Ilosvay-reagent to the tubes (nitrite A and B reagent). Mix the contents of the tube. The presence of nitrite in the broth is demonstrated by a cherry red color within 30 sec. There are two possibilities if there isn’t red coloration: either ammonia oxidation has not taken place (effect of the inhibitor), or nitrite was completely oxidized to nitrate. In order to be able to distinguish between these two options, add a small amount of zinc to the tubes (max. 5 mg/ml). If nitrate has formed during nitrification, zinc would reduce it back to nitrite, and the Griess-Ilosvay-reagent will form a red coloration. If there is still no color change after the addition of zinc, add Nessler-reagent to the remaining broth. If a yellowish brown color develops it indicates the unchanged ammonium ions. If there is no red coloration in the nitrite-containing broth, then nitrite oxidation was complete. To prove this assumption, perform the zinc reduction test.

Nutrient media The presence of which compound have you demonstrated?

What kind of oxidation has taken place?

NH4+ NO2

- NO3- Ammonia Nitrite

Ammonia Ammonia + ATU Nitrite Nitrite+ ATU

Fig.11.3. Evaluation of experiments used for the demonstration of nitrification D. DEMONSTRATION OF DISSIMILATIVE NITRATE REDUCTION During the dissimilative reduction of nitrate or nitrite, the end products are nitrite, dinitrogen-oxide, nitrogen gas or ammonia. The process is anaerobic and takes place in compacted, water saturated soils; river or lake sediments; inside the gastrointestinal tract of higher organisms, etc. When a gaseous substance (N2O, NO, N2) is produced, the process is called denitrification. This process plays a very important role in the nitrogen equilibrium and self purification of soil and water. From the biochemical point of view, this process is

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nitrate reduction: the utilization of nitrate as electron acceptor for the biological oxidation of different organic and inorganic (e.g. H2S) substances.

Sample: own unidentified strain, soil suspension Materials: Nitrate broth containing Durham test tubes, zinc powder, Nessler-

reagent, Griess-Ilosvay-reagent (nitrite reagent)

Exercise: Inoculate the nitrate media with 1 ml soil suspension. Incubate the samples at 28°C for a week and evaluate the results for the presence of the following products: Nitrite (NO2

-): Griess-Ilosvay reagent Nitrogen gas (N2): Bubbles inside the Durham tubes Ammonia: Nessler-reagent E. EVALUATION OF RESULTS OF WATER ANALYSIS

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PRACTICAL XII

FERMENTATION PROCESSES IN BIOTECHNOLOGY Biotechnology in the classical sense is defined as an industrial production technique, where some living organism or components of them (e.g. enzymes) carry out the production of the required product. Technologies based on “microbial fermentations” can be defined as traditional biotechnological techniques. Through in microbial fermentations, microorganisms in an artificial environment in fermentors (at aerobic or anaerobic conditions) produce materials (primary or secondary metabolites), such as antibiotics, citric acid, alcohol. Nowadays the use of recombinant DNA techniques made it possible to produce several compounds (e.g. insuline, growth factors, interferon etc.) by genetically modified living organisms. (Table 12.1). These organisms can be plants and animals or their cell cultures besides microorganisms.

Metabolites Microorganisms Products intended for industrial use

Ethanol (from glucose) Ethanol (from lactose) Acetone and butanol 2,3-butanediol Enzymes (amylase, protease etc.)

Saccharomyces cerevisiae Kluyveromyces fragilis Clostridium acetobutylicum Enterobacter, Serratia Aspergillus. Bacillus, Mucor, Trichoderma

Products intended for agricultural use Gibberellins Gibberella fujikuori

Food additives Amino acids (lysine etc.) Organic acids (citric acid) Nucleotides Vitamins Polysaccharides

Corynebacterium glutamicum Aspergillus niger Corynebacterium glutamicum Ashbya, Eremothecium, Blakeslea Xanthomonas

Products intended for medical use Antibiotics Alkaloids Steroids Insulin, human growth factor, interferon, somatostatin, etc.

Penicillium, Streptomyces, Bacillus Claviceps purpurea Rhizopus, Arthrobacter Escherichia coli, Saccharomyces cerevisiae and other organisms (recombinant DNA techniques)

Products used as fuels Hidrogen Methane Ethanol

Photosynthetic microorganisms Methanobacterium Zymomonas, Thermoanaerobacter

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Microbial metabolites can be divided into two groups: primary and secondary metabolites. The primary metabolites essential for cellular life and reproduction are synthesized during the growth phase (trophophase). These substances include: amino acids, nucleotides, fermentation end products (e.g. ethanol and organic acids). The secondary metabolites however are produced in the idiophase, following the growth phase. The cell no longer replicates and grows through changes that can be accounted for lack of nutrients, accumulation of toxic substances, lack of oxygen or other essential compounds needed for the synthesis of cell components, growth and replication. These substances accumulate within the nutrient medium, they differ in their chemical structure and physiological impact (e.g. antibiotics, micotoxins). A.1. CITRIC ACID PRODUCTION IN SHAKEN CULTURE Nowadays, citric acid (2-hydroxil-propane-1,2,3-tricarbolicacid) is produced microbiologically by Aspergillus niger and Aspergillus wentii. The most important carbon source for citric acid production is glucose. Some of the glucose is used up during culturing for mycelium growth in the trophophase, while glucose is used for citric acid production during the idiophase. Exercise: Take Aspergillus niger spores from an agar slant into sterile distilled water using an inoculating loop. Set spore concentrations of the suspensions at 20,000 and 80,000 spores/ml using the Bürker chamber. Add 1 ml of each spore suspension into separate Erlenmeyer flasks containing 50 ml nutrient media for citric acid production. Shake the flasks at 37°C for a week at 270 rpm. Demonstrate the citric acid production by thin layer chromatoghraphy (TLC).

Strain: Aspergillus niger Materials: Bürker chamber, thin layer chromatographic sheets, microcapillaries,

forceps, hair dryer, nutrient media for citric acid production

Fig. 12. 1. Bürker chamber Immobilised cell culture – fermentation of alcohol Alcohol production, in the industry, by Saccharomyces cerevisiae cells. Advantages of immobilized cell technique are:

1. Higher cell density 2. microbial biomass can be used several times 3. continuous fermentation 4. less possibility of contamination 5. product can be easily purified

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Techniques:

Binding to a carrier

Fig. 12. 2. Immobilized cell techniques Binding to a carrier is based on the technique that the cells or enzymes are bound to a solid carrier with ionic or covalent bonds. Carriers can be not water soluble polysaccharides (cellulose, dextrane, agaroise), proteins (gelatine, albumine), synthetic polymers (ion exchange resins, polyvinyl chloride) and inorganic compounds (quartz). At the cross-binding method, 2 or more function group reagents (glutarealdehide) will react with the cells. At the capturing method, cells will bind into a polymer material (alginate, polyacrylamide). Exercise: Saccharomyces cerevisiae cells will be immobilized in Ca-alginate gel and will ferment alcohol from glucose and malate. 50 ml Saccharomyces cerevisiae cell suspension (about 25 g wet cell biomass) sterily mixed with 4% alginate solution, then this will be dropped into 0.15M CaCl2 (pH6-8) solution at 37°C. Solidify for 1 hr at 20-22°C, then stabilize overnight at 4°C. Solidifying solution is changed to glucose solution next morning, fermentation at 28°C. Next week, alcohol is detected with H2SO4 diluted potassium/dichromate. Strain: Saccharomyces cerevisiae

Cross binding

Capturing

Covalent bond adsorption

=cell or enzyme