microbiology lab manual'15 batch.pdf

7/26/2019 Microbiology lab manual'15 batch.pdf

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KARPAGAM UNIVERSITYKarpagam Academy of Higher Education

(Deemed to be University, Established under Section 3 of UGC Act 1956)COIMBATORE-641 021.

Faculty of Engineering

Department of Biotechnology

15BTBT312

MICROBIOLOGYLAB

PREPARED BY

Dr. R. Thilagavathi & Ms. P. Sandhya


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(Deemed to be University, Established under Section 3 of UGC Act 1956)

COIMBATORE-641 021.

MICROBIOLOGY

LABORATORY RECORD BOOK

Name : ..

Register No. :


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(Deemed to be University, Established under Section 3 of UGC Act 1956)

COIMBATORE- 641 021.

This is to Certify that this.......

.... (Lab Name) record work done

by Mr./Ms/Mrs.......

for the course B.Tech...

(branch) during..(year/semester) of

Academic year 20162017 is bonaf ide.

Facultyin-charge H.O.D

REGISTER No. .....

This record is Submitted for.... Semester

B.Tech, PracticalExamination of Karpagam University conducted on .....

I nternal Examiner External Examiner


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EXP

NO.

DATENAME OF THE

EXPERIMENT

PAGE

NO.

MARKS

TOTAL

(100)

FACULTY

SIGN

PREP(30)

OBS(30)

REC(30)

VIVA(10)

1

Laboratory safety and aseptic

techniques

2 Microscopylight microscopy

3

Culture mediaTypes,

Preparation of nutrient broth and

nutrient agar

4

Culturing of microorganismsin

Broth and in plates

(Spread plate, Pour plate, Streak

plate)

5

Staining techniques

Motility test

6 Quantitation of microorganisms

7

Chemical control of

microorganisms

Antibiotic sensitivity assay

8 Bacterial growth curve

9

Effect of different parameters on

bacterial growth

(Temperature and UV

irradiation)

INDEX

AVG:


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EXP. NO: 1 LABORATORY SAFETY AND ASEPTIC TECHNIQUES

DATE:

AIM:

To understand the lab safety guidelines, bio-safety rules and aseptic techniques to be followed

in the microbiology lab.

SAFETY GUIDELINES:

Safety in a microbiology laboratory is important in the prevention of infection that might be

caused by the microorganisms being studied. This laboratory does not require the use of virulent

human pathogens. However, many types of microorganisms are potentiallypathogenic. This

means that, although they would not cause disease in a normal healthy host, they might possibly do

so if a large enough quantity of the microbes came into contact with a compromised host, such as by

wounds and cuts.

In addition to microorganisms, there are some chemicals used in this laboratory that

are potentially harmful. Many procedures involve glasswares, open flames, and sharp objects that

can cause damage if used improperly.

Specific lab safety guidelines are designed to address each of these potential routes of

exposure.

Microbiology Laboratory Safety Rules

1. Never work alone in the laboratory without permission and prior knowledge of the instructor.

2. Do not engage in rowdy, playful, or unprofessional activities in the laboratory.

3. Work surfaces must be disinfected at the beginning and at the end of every laboratory period.

4. All students must wash their hands at the beginning of the lab and at the end of every laboratory

period.

5. No eating or drinking is permitted in the laboratory. No open food or beverage can be taken into

the laboratory.

6. Lab benches are to be kept free of extraneous items while conducting experiments. This includes

personal items such as backpacks, cell phones, and unnecessary books.

7. Students must wear closed-toe shoes that cover the top of the foot, and appropriate clothing, at all

times in the laboratory.


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8. Students must wear gloves when handling microorganisms. Wear lab aprons or lab coats as

advised by your instructor. Wear safety glasses when handling bacterial broth cultures, doing

Gram stains, and as otherwise advised by your instructor.

9. Keep hands away from your face, eyes, and mouth when working with chemicals or

microorganisms. This includes not applying cosmetics, not adjusting contact lenses, and not

biting your finger nails.

10.If any chemicals or other agents splash into your eyes, immediately go to the nearest sink and

flush your eyes with water.

11.Take special precautions when working with open flames. Loose hair, clothing, dangling jewelry,

and nearby paper must be secured. Do not leave a heat source (hot plate or Bunsen burner)

unattended. Keep containers of alcohol, acetone, or other flammable liquids at a safe distance

from flames.

12.Report ANY and ALL accidents, spills, BREAKAGES, or injuries to the instructor, no matter

how trivial they appear.

13.Any pregnant or immunocompromised student must notify the instructor of the course. A

pregnant student is required to wear safety glasses and 2 sets of examination gloves when

handling any bacterial broths or cultures.

14.Do not remove cultures, reagents, or other materials from the laboratory unless specific

permission from the instructor has been granted.

15.Do not use any lab equipment without instruction and authorization from the instructor. Report

any damaged or broken equipment to your instructor immediately.

16.Students must assume that all of the organisms that they work with in this laboratory are potential

pathogens (disease-producing microbes). However, this laboratory does not require the use of any

highly infectious human pathogens. Bacterial culture material used in this lab can include the

following organisms: Bacillus subtilis, Corynebacterium xerosis, Enterobacter cloacae,

Escherichia coli, Klebsiella pneumoniae, Lactobacillus casei, Micrococcus luteus, Proteus

vulgaris, Pseudomonoas aeruginosa, Salmonella typhimurium, Serratia marcescens,

Staphylococcus aureus, Staphylococcus epidermis, and Streptococcus mutans.


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ASEPTIC TECHNIQUES

Only non-pathogenic cultures should be used in schools obtained from a recognised educational

supplier. Sterile equipment and media should be used in the transfer and culture of microorganisms.

Aseptic technique should be observed whenever microorganisms are transferred from one container

to another.

It is wise to treat all cultures as potentially pathogenic, because cultures may have been

contaminated, and because mutations to disease-causing forms may occur. The aseptic techniques

described here control the opportunities for contamination of cultures by microorganisms from the

environment, or contamination of the environment by the microorganisms being handled.

There are some general rules to follow for any aseptic technique.

1.

Close windows and doors to reduce draughts and prevent sudden movements which might

disturb the air.

2.

Make transfers over a disinfected surface. Ethanol disinfection is recommended because of its

rapid action. If the bench surface is difficult to clean, cover the bench with a sheet of tough

material which is more easily disinfected.

3. Start the operations only when all apparatus and materials are within immediate reach.

4. Complete all operations as quickly as possible, but without any hurry.

5.

Vessels must be open for the minimum amount of time possible.

6. While vessels are open, all work must be done close to a Bunsen burner flame where air

currents are drawn upwards.

7. On opening a test tube or bottle, the neck must be immediately warmed by flaming (see

below) with the vessel held as near to horizontal as possible and so that any movement of air

is outwards from the vessel.

8. During manipulations involving a Petri dish, limit exposure of the sterile inner surfaces to

contamination from the air.

9. The parts of sterile pipettes which will be put into cultures or sterile vessels must not be

touched or allowed to come into contact with other non-sterile surfaces, such as clothing, the

surface of the working area, or the outside of bottles/ test tubes.

10. All items which come into contact with microorganisms must be sterilised before and after

each such exposure. This could be either by the technical team preparing for and clearing up


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after a piece of practical work (for example, in the case of glassware to be used), or by the

worker during the course of the practical (for example, in flaming a wire loop).

Wire loops

For transferring fungal cultures which grow by producing a mycelium of hyphae, an inoculation wirewith the end bent into a small hookis better than a loop. Use the hook to gouge into the agar at the

edge of the culture and pick up a small piece of agar plus hyphae. Transfer this to agar plate or slope,

and invert the piece of fungus agar so that the fungus is in contact with the agar in the dish or tube.

Ensure that the culture adheres firmly to the new agar. You may decide not to invert agar plates

immediately in case the transferred culture falls off the agar.

Pipettes

A leaking pipette is caused either by a faulty or ill-fitting teat, or by fibres from the cotton wool plugbetween the teat and the pipette.

A dropping (Pasteur) pipette can be converted to deliver measured volumes by attaching it by rubber

tubing to a non-sterile syringe barrel.

Cotton wool plugs

Cotton wool stoppers are easier for students to handle than screw caps this makes complex

manipulations more straightforward.

If a plug accidentally catches fire, douse the flames immediately by covering with a dry cloth, not byblowing or soaking in water.

Inoculating agar plates, slopes and cultures

aCarry out the transfer of cultures as quickly as possible, with tubes and plates open to the air for the

minimum length of time.

bNormal practice is to open agar plates away from the body and without removing the lid

completely from the base.

cWhen the lid of the Petri dish is removed for longer periods than normal, work very close to theBunsen burner flame to reduce the chances of contamination.

dIf you experience frequent contamination of plates with fungal spores, reduce the chance of

draughts further, and consider inoculating plates from below with the agar surface facing downwards.

In this way there is perhaps less chance of spores settling onto the plate from the air.


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Using a wire loop

a Clean the loop by heating to red hot as described below, allow to cool, then reshape with forceps

before beginning again. Do not use your fingers because of the possibility of puncturing your skin.

bHold the handle of the wire loop close to the top, as you would hold a pen, at an angle that isalmost vertical. This leaves the little finger free to take hold of the screw cap/ cotton wool plug of the

bottle/ test tube. It also ensures that any liquid culture on the loop will run down into the flame.

cSterilize the wire loop by heating to red hot in a roaring blue Bunsen burner flame before and after

use. This ensures that contaminating bacterial spores are destroyed.

dThe flaming procedure should heat the tip of the loop gradually. This is because after use it will

contain culture, which may splutter on rapid heating and possibly release small particles of culture,

forming an aerosol.

i Position the handle end of the wire in the light blue cone of the flame. This is the coolest area of

the flame.

ii Draw the rest of the wire upwards slowly into the hottest region of the flameimmediately above

the blue cone.

iii Hold there until it is red hot.

iv Ensure the full length of the wire receives adequate heating.

v Allow to cool for a few seconds in the air, then use immediately.

vi Do not put the loop down, or wave it around.

viiRe-sterilise the loop immediately after use.

Using a pipette

Sterile graduated or dropping (Pasteur) pipettes are used to transfer cultures, sterile media and sterile

solutions.


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aRemove the pipette from its container/ wrapper by the end containing a cotton wool plug, taking

care to touch as little of the pipette as you need to take a firm hold.

bFit the teat. It is sometimes helpful to dip the teat first in sterile liquid to lubricate it.

cHold the pipette barrel as you would a pen, but do not grasp the teat. This leaves your little fingerfree to take hold of the cap/ cotton wool plug of a bottle/ test tube and your thumb free to control the

teat.

dDepress the teat cautiously and take up an amount of fluid which is adequate for the amount

required, but does not reach and wet the cotton wool plug. Squeezing the teat with the pipette tip

beneath the liquid surface introduces air bubbles which may cause spitting and, consequently,

aerosol formation. Avoid this by squeezing the teat before placing the tip into the liquid. Then gently

release the pressure until the required amount of liquid is drawn up, and lift the pipette tip out of the

liquid.

eReturn any excess gently.

fImmediately after use put the contaminated pipette into a nearby discard pot of disinfectant.

g Remove the teat only once the pipette is within the discard pot otherwise drops of culture will

contaminate the working surface.

Flaming the neck of bottles and test tubes


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This ensures that no microorganisms enter the mouth of the vessel to contaminate the culture or the

medium. Passing the mouth of the bottle through a flame produces a convection current away from

the opening, and helps to prevent contamination. The hot part of the flame is above the inner bright

blue cone and the vessel needs to be moved through the flame, not held in place.

aLoosen the cap of the bottle so that it can be removed easily.

bLift the bottle/ test tube with your left hand.

cRemove the cap/ cotton wool plug of the bottle/ test tube with the little finger curled towards the

palm of your right hand. (Turn the bottle, not the cap.)

dDo not put down the cap/ cotton wool plug.

eFlame the neck of the bottle/ test tube by passing the neck forwards and back through a hot Bunsen

burner flame.

fAfter carrying out the procedure required, for example, withdrawing culture, replace the cap/ cotton

wool plug on the bottle/ test tube using your little finger. Take care! The bottle will be hot. (Turn the

bottle, not the cap.)

gIf cotton wool plugs have partly lost their shape, they can be more easily guided back into the neck

of the vessel by slowly twisting the mouth of the vessel as the plug is pushed down.

Disinfecting surfaces

aFor technicians, ethanol disinfection is recommended because of its rapid action (around 5

minutes). Technicians will be more experienced and able to deal with the associated fire hazards ofworking with ethanol.

bFor disinfection by students, 1% Virkon solution is safer and cheaper, but the surface must be left

wet for 10 minutes.


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STERILIZATION TECHNIQUES

Sterilization is the killing or removal of all microorganisms, including bacterial spores which

are highly resistant. Sterilization is an absolute term, i.e. the article must be sterile meaning the

absence of all microorganisms.Disinfection is the killing of many, but not all microorganisms. It is a

process of reduction of number of contaminating organisms to a level that cannot cause infection, i.e.

pathogens must be killed. Some organisms and bacterial spores may survive.Disinfectants are

chemicals that are used for disinfection. Disinfectants should be used only on inanimate

objects.Antiseptics are mild forms of disinfectants that are used externally on living tissues to kill

microorganisms, e.g. on the surface of skin and mucous membranes.

Uses of Sterilization:

1. Sterilization for Surgical Procedures: Gloves, aprons, surgical instruments, syringes etc. are to besterilized.

2. Sterilization in Microbiological works like preparation of culture media, reagents and equipments

where a sterile condition is to be maintained.

Classification of Methods:

Sterilization and disinfection are done by :

(A). Physical Agents

1. Heat

2. Radiation

3. Filtration

(B). Chemical Agents

In practice, certain methods are placed under sterilization which in fact do not fulfill the definition of

sterilization such as boiling for 1/2 hr and pasteurization which will not kill spores.

STERILIZATION BY HEAT

Heat is most effective and a rapid method of sterilization and disinfection. Excessive heat acts by

coagulation of cell proteins. Less heat interferes metabolic reactions. Sterilization occurs by heating

above 100C which ensure lolling of bacterial spores. Sterilization by hot air in hot air oven and

sterilization by autoclaving are the two most common method used in the laboratory.

Types of Heat :

A. Sterilization by moist heat

B. Sterilization by dry heat


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MOIST HEAT:

Moist heat acts by denaturation and coagulation of protein, breakage of DNA strands, and loss of

functional integrity of cell membrane.

Sterilization at 100C:

1. Boiling. Boiling at 100C for 30 minutes is done in a water bath. Syringes, rubber goods and

surgical instruments may be sterilized by this method. All bacteria and certain spores are killed. It

leads to disinfection.

2. Steaming. Steam (100C) is more effective than dry heat at the same temperature as: (a) Bacteria

are more susceptible to moist heat, (b) Steam has more penetrating power, and (c) Steam has more

sterilizing power as more heat is given up during condensation.

Steam Sterilizer. It works at 100C under normal atmospheric pressure i.e. without extra pressure. It

is ideally suitable for sterilizing media which may be damaged at a temperature higher than 100C.

It is a metallic vessel having 2 perforated diaphragms (Shelves), one above boiling water, and the

other about 4" above the floor. Water is boiled by electricity, gas or stove. Steam passes up. There is

a small opening on the roof of the instrument for the escape of steam. Sterilization is done by two

methods :

(a) Single Exposure for 11/2 hours. It leads to disinfection.

(b) Tyndallization (Fractional Sterilization). Heat labile media like those containing sugar, milk,

gelatin can be sterilized by this method. Steaming at 100C is done in steam sterilizer for 20 minutes

followed by incubation at 37C overnight. This procedure is repeated for another 2 successive days.

That is 'steaming' is done for 3 successive days. Spores, if any, germinate to vegetative bacteria

during incubation and are destroyed during steaming on second and third day. It leads to sterilization.

Sterilization by Dry Heat:

Mechanisms.(1) Protein denaturation, (2) Oxidative damage, (3) Toxic effect of elevated electrolyte

(in absence of water).

Dry heat at 160C (holding temperature for one hour is required to kill the most resistant spores). The

articles remain dry. It is unsuitable for clothing which may be spoiled.

1. Red Heat. Wire loops used in microbiology laboratory are sterilized by heating to 'red' in bunsen

burner or spirit lamp flame. Temperature is above 100C. It leads to sterilization.

2. Flaming. The article is passed through flame without allowing it to become red hot, e.g. scalpel.

Temperature is not high to cause sterilization.


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3. Incineration : It is an excellent method for destroying the materials rapidly. Eg: Animal dead

bodies, beddings, pathologic materials.

STERILIZATION BY AUTOCLAVE

Sterilization above 100C: Autoclaving

Autoclaving is one of the most common methods of sterilization. Principle: In this method

sterilization is done by steam under pressure. Steaming at temperature higher than 100C is used in

autoclaving. The temperature of boiling depends on the surrounding atmospheric pressure. A higher

temperature of steaming is obtained by employing a higher pressure. When the autoclave is closed

and made air-tight, and water starts boiling, the inside pressures increases and now the water boils

above 100C. At 15 ib per sq. inch pressure, 121C temperatures is obtained. This is kept for 15

minutes for sterilization to kill spores. It works like a pressure cooker.'Sterilization holding time' is

the time for which the entire load in the autoclave requires to be exposed.Autoclave is a metallic

cylindrical vessel. On the lid, there are : (1) A gauge for indicating the pressure, (2) A safety valve,

which can be set to blow off at any desired pressure, and (3) A stopcock to release the pressure. It is

provided with a perforated diaphragm. Water is placed below the diaphragm and heated from below

by electricity, gas or stove. Working of Autoclave. (a) Place materials inside, (b) Close the lid. Leave

stopcock open, (c) Set the safety valve at the desired pressure, (d) Heat the autoclave. Air is forced

out and eventually steam ensures out through the tap, (e) close the tap. The inside pressure now rises

until it reaches the set level (i.e. 15 Win), when the safety valve opens and the excess steam escapes,

(f) Keep it for 15 minutes (holding time), (g) Stop heating, (h) Cool the autoclave below 100C, (i)

Open the stopcock slowly to allow air to enter the autoclave002E

Checking of Autoclave for Efficiency.Methods :

(i) Spores of Bacillus stearothermophilus are used. Spores withstand 121C heat for up to 12 min.

Strips containing this bacteria are included with the material being autoclaved. Strips are cultured

between 50C and 60C for surviving spores. If the spores are killed the autoclave is functioning

properly.

(ii)Automatic Monitoring System.

STERILIZATION BY HOT AIR OVEN

Hot Air Oven (Sterilizer). It Is one of the most common method used for sterilization. Glass wares,

swab sticks, all-glass syringes, powder and oily substances are sterilized in hot air oven. For


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sterilization, a temperature of 160C is maintained (holding) for one hour. Spores are killed at this

temperature. It leads to sterilization.

Hot Air Oven is an apparatus with double metallic walls and a door. There is an air space between

these walls. The apparatus is heated by electricity or gas at the bottom. On heating, the air at the

bottom becomes hot and passes between the two walls from below upwards, and then passes in the

inner chamber through the holes on Me top of the apparatus. A thermostat is fitted to maintain a

constant temperature of 160C.

STERILIZATION BY RADIATION

ULTRAVIOLET LIGHT:

This is commonly employed to aid the sterilization of air and surfaces in the processing

environment. UV light penetrates clean air and pure water. When UV light passes through matter,

energy is liberated to the orbital electrons within the constituent atom. This absorbed energy causes

highly energized state of atoms and alters their reactivity. UV lamps are used for their germicidal

effects on the surfaces or their penetrating effect through clean air and water

IONIZING RADIATIONS:

Ionizing radiations are high energy radiations emitted from radioactive isotopes such as Cobalt 60 (

rays) or produced by mechanical acceleration of electrons to verify velocity and energy ( rays).

Ionizing radiations destroy the microorganism by stopping the reproduction as a result of lethal

mutations. These ionizing radiations are particularly used for sterilization of medical classic devices.

Vitamins, antibiotics, hormones in dry state are also sterilized by this method.

STERILIZATION BY FILTRATION:

It is the method used for the removal of particles including microorganisms from solutions and gases

without the application of heat. Membrane filters function primarilyby screening particles from a

solution or gas thus retaining them on filter surface. Membrane filters also function in some instances

by electrostatic attraction. This would apply particularly to the filtration of dry gases.

LAMINAR FLOW CHAMBERS:

They are used to maintain specific aseptic work area. They are of two types:


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Horizontal flow type

Vertical flow type

It consists of a HEPA filter (High Efficiency Particulate Air) to which air is forced to move at a

slow rate of 0.45 m/sec across the working space. Usually HEPA filters are capable of removing

99.97 to 99.99% of particles and bacteria of 0.3m and above. A pre filter which is made of a

glass fiber that has the ability to retain 99% of particles of size 5m or above is included in the

system. In such cabinets, the risk of contaminations during aseptic processing condition is very

low. To work with pathogenic microorganisms, cabinet with vertical flow is necessary to protect

the operator.

CHEMICAL METHODS OF STERILIZATION:

ETHYLENE OXIDE:

It penetrates rapidly through materials such as plastics, powders and paper boards. It exerts its effect

upon the microorganism by alkylating the essential metabolites affecting primarily the reproductive

process. It has extensive application in sterilizing plastic materials, rubber boots and delicate optic

instruments.

- PROPIOLACTONE:

These are bacteriocidal used agents against wide variety of microorganisms at relatively low

concentration. It is an alkylating agent and has mode of action similar to that of ethylene oxide. This

is used for sterilization of large surfaces such as an entire room.

PHENOL:

Phenol is a powerful bacteriocidal agent used for disinfection. The mode of action may be self-

precipitation, inactivation of enzymes present in the membrane. Eg: Lysol, Cresol and thymol. These

compounds are used for sterilizing surgical instruments.

ALCOHOLS:

70% ethanol and isopropyl alcohol are used for disinfections. It denatures the cellular proteins and

make them inactive.


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HALOGENS:

The most widely used halogens for disinfection are iodine and chlorine compounds. They are used as

disinfectants for skin against both Gram Positive and Gram negative bacteria. It acts by oxidizing the

proteins.

Cl2+ H2O HCl + HOCl

HOCl HCl + [O]

HEAVY METALS AND THEIR COMPOUNDS:

Mercuric Chloride and silver nitrate prevent the growth of most bacteria. Copper salts give fungicidal

effect. These heavy metals combine with enzymes and make them inactive, disrupting the

metabolism of microbes.

SH S

Enzyme + HgCl2 Enzyme Hg + 2 HCl

SH S

ALDEHYDES:

5-10% formaldehyde solution kills most bacteria. It also acts as bactericidal, sporicidal and are lethal

to viruses. It combines with Organic Nitrogen present in Nucleic acids and Proteins. Glutaraldehyde

is an excellent disinfectant for tuberculbacilli, fungi and viruses. It is used in operation theatres, face

mask, wounds and viral inoculating chambers.

DYES:

Aniline dyes like Malachite green, brilliant green and crystal violet are active against G+ve bacteria

and are also used as antiseptic for skin and wounds. Acridine dyes are also used as bacteristatic agent

ACIDS:

Inorganic acids, boric acid act as a bacteriocidal and anti-fungal agent. Formic acid is a powerful

germicide. Organic acid, benzoic acid and salicylic acid are anti-fungal and bacteriostatic agents


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RESULT:


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VIVA QUESTIONS:

1. What are the basic routes of exposure to microorganisms?

2. How will you avoid the exposure to microorganisms?

3. How will you dispose the microbiological waste?

4. Why the aseptic technique needs to be followed?

5. If any accidents take place what will you do?


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EXPERIMENT NO: 2

MICROSCOPYLIGHT MICROSCOPY


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EXP. NO: 2 MICROSCOPYLIGHT MICROSCOPY

DATE:

AIM:

To study the different parts of the light microscope and the principles of microscopy.

To prepare a wet mount.

PRINCIPLE:

The purpose of a microscope is to magnify an object, which often cannot be seen

without a microscope, so that it can be seen with the naked eye. The light microscope is an

important tool in the study of microorganisms. The compound light microscope uses visible

light to directly illuminate specimens in a two lens system, resulting in the illuminated specimen

appearing dark against a bright background. The two lenses present in a compound microscope

are the ocular lens in the eyepiece and the objective lens located in the revolving nosepiece.

Compound light microscopes typically have the following components

1. Illuminator:the light source in the base of the microscope.

2. Abbe Condensor: a two lens system that collects and concentrates light from the

illuminatorand directs it to the iris diaphragm.

3. Iris Diaphragm:regulates the amount of light entering the lens system.

4.

Mechanical Stage:a platform used to place the slide on which has a hole in the center to

letlight from the illuminator pass through. Often contains stage clips to hold the slide in

place.

5. Body tube: Houses the lens system that magnifies the specimens.

6. Upper end of the body tube --Oculars/Eye pieces:what you view through.

7. Lower end of the body tube --Nose-piece:revolves and contains the objectives


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Figure 1: Light Microscope.

PRINCIPLE

Basically, a light microscope magnifies small objects and makes them visible. The

science of microscopy is based on the following concepts and principles:

Magnificationis simply the enlargement of the specimen. In a compound lens system, each

lens sequentially enlarges or magnifies the specimen. The objective lens magnifies the specimen,

producing a real image that is then magnified by the ocular lens resulting in the final image. The

total magnificationcan be calculated by multiplying the objective lens value by the ocular

lensvalue.

Resolving power is the ability of a lens to show two adjacent objects as discrete

entities. In general, the shorter the wavelength of light, the better the resolution, which is why a blue

filter is usually connected to the condenser to produce short light waves for optimum resolution.

Resolving power is also dependent on the refractive index or the bending power of light. Because air

has a lower refractive index than glass, light waves have a tendency to bend and scatter as

they pass through the air from the glass slide to the objective lens. Addition of immersion oil, which

has the same refractive index as glass, diminishes the loss of refracted light and improves resolution.


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Contrastis the ability to distinguish an object from its background. Since most microbes are

relatively transparent when viewed under a standard light microscope they are difficult to identify.

Using a stain (labs 2-5) that will bind to the microorganism and not the glass slide

dramatically enhances their contrast enabling them to be observed more clearly.

Depth-of-focus is the thickness of the sample that appears in focus at a particular

magnification. As the magnification increases the depth-of-focus decreases, or the slice of

the sample thatappears in focus gets thinner. Many of the newer compound microscopes are par

focal, which means that if one objective lens has the object in focus, and you go to the next objective

lens, only minor adjustment (fine focus) is needed to bring the image back into focus. This is due to

the fact that as you increase the magnification, and thus the slice of the sample that appears

in focus becomes thinner, the correct plane-of-focus will always be within the depth of

focus of the previous objective. After you get the sample into focus at scanning or low power using

the course adjustment knob, you should only have to use the fine focus knob at the higher

magnifications.

Field-of-Viewis the area of the slide that you are observing through the microscope.

As you increase the magnification the actual area of the slide that you are looking at is getting

smaller. You can think of the field-of-view as a dartboard. At low magnification you are able to see

the entire dartboard, but as you increase the magnification you are only observing the bulls-

eye, a much smaller portion of the dartboard.

These microscopes are also par central,which refers to the ability to keep an object in the

middle of your field-of-view when changing from one objective to another. It is useful to remember

this as you are increasing magnification. Always keeping your sample in the center of your field-of-

view will avoid unnecessary searching of the slide for your sample.

Working distance is the distance between the objective and the slide. As you increase

magnification (by using more powerful objective lenses) the working distance decreases. So much so

that by the time you are using the oil-immersion objective (100X) the objective is almost

touching the slide, allowing the immersion oil to connect the slide and objective. It is important to

consider working distance in a number of applications, but practically there are two reasons you

should be aware of your working distance. The first is so that you do not inadvertently push the

objective through the slide, causing damage to the objective and your sample slide. The second isto

estimate whether you are in the correct plane-of-focus.


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Materials:

Microscope

Newsprint

Stage micrometer

Slides

Coverslips

Transfer pipettes

Prepared slides of bacteria

Hay infusion

Protoslow

Immersion oil

Lens paper

PROCEDURE:

1.Place a piece of newsprint on a microscope slide and cover with a coverslip. ALWAYS USE A

COVERSLIP!

2.Turn the microscope on and set the light source on its highest setting.

3.Use the coarse adjustment knob to obtain maximum working distance.

4.Place the slide on the stage. The slide should fit into the slide holder but is not placed under the

slide holder. Use the stage adjustment knob to move the slide the edge of the coverslip bisects the

hole in the stage.

5.Rotate the scanning objective (4X) into place.

6.Use the coarse adjustment knob to obtain the minimum working distance. Develop the habit of

watching this process to be sure the objective does not crash into the slide.

7.Look through the oculars. Adjust the light with the iris diaphragm lever on the condenser if

necessary. Slowly turn the coarse adjustment knob until the edge of the coverslip comes into focus.

Use the fine adjustment knob to sharpen the focus.8.Use the stage adjustment knob to locate the letter e in the newsprint. Note the orientation of the

letter e in the newsprint.

9.Rotate a higher power objective (10X) into place. Use the fine adjustment knob to sharpen the

focus. Do not use the coarse adjustment knob. Adjust the light using the iris diaphragm lever if


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necessary. The image is now magnified 100X (10X ocular x 10X objective = 100X magnification).

Draw the letter e as it appears in the microscope on the lab report sheet.

10.Place a stage micrometer on the stage and determine the diameter of the field of view for all four

objectives. The micrometer is 2 mm in length. The ruler is divided into tenths. Record the distances

on the lab report sheets.

11.When using the high power objective (100X) use the following procedure. Rotate the turret

halfway between the 40X and 100X objective. Place a drop of immersion oil on the slide and rotate

the oil immersion objective (100X) into place. The objective should be immersed in the oil on the

slide. Use the fine adjustment knob to sharpen the focus. Adjust the light using the iris diaphragm

lever if necessary. Never use the coarse adjustment knob with high power.

12.Place a drop of water from the hay infusion on a microscope slide. Cover with a coverslip and

view under all four objectives. Sketch two (2) of the organisms at 400X magnification.

13.Obtain a prepared slide for two bacterial species. View slides under the 1000X objective and

sketch the bacteria. Dont forget the immersion oil!

14.When you are finished with the microscope clean the microscope, as described below, and return

it to storage.

PROCEDURE FOR CLEANING A MICROSCOPE:

1.Turn off the light and unplug the cord. Store the cord appropriately.

2.Using the coarse adjustment knob to obtain maximum working distance and

remove the slide from the stage.

3.Using lens paper clean all the lenses starting with the cleanest firstoculars,

4X through 100X objectives.

4.Clean any oil off of the stage using Kimwipes or paper towels.

5.Rotate the scanning objective into place. Use the coarse adjustment knob to

obtain minimum working distance.

6.Return the microscope to the appropriate storage area.


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OBSERVATION:

RESULT:


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VIVA QUEST IONS:

1. State the purpose of each of the following microscope components:

a. Condenser

b. Fine-adjustment knob

c. Coarse-adjustment knob

d. Iris Diaphragm


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e. Mechanical stage control

2. What is the purpose of adding immersion oil when using the 100X objective?

3. If the ocular lens has a magnification of 10X and the objective lens has a magnification

of40X, what is the total magnification?


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4. What is refractive index?

5. Name the types of microscopy techniques


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EXPERIMENT NO: 3

CULTURE MEDIATYPES, PREPARATION OF

NUTRIENT BROTH AND NUTRIENT AGAR


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EXP. NO: 3 CULTURE MEDIATYPES, PREPARATION OF

DATE: NUTRIENT BROTH AND NUTRIENT AGAR

AIM:

To study the different types of culture media, preparation of nutrient broth and nutrient agar.

PRINCIPLE:

All microorganisms have certain nutritional requirements for their growth. All organisms

require an energy source, an electron source, carbon, nitrogen, oxygen, sulphur, phosphorous, trace

elements and water. To study the morphological, cultural and biochemical characteristics of various

organisms, it is essential to culture the media. Medium is a substance that provides nutrients for

growth and multiplication of the organism. In addition to nutrients, microorganisms need various

environmental factors such as temperature, pH etc. Many special purpose media are needed to

facilitate recognition, enumeration and isolation of certain traits of bacteria. Based on the accuracy of

nutritional values, media are of two types, Complex or Chemically undefined media which is

composed of compounds whose exact chemical composition are not known. Undefined or synthetic

media has known quantity of specific compounds.

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, gelatin], 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 (0.1-0.2% agar)can also be used in fermentation studies, in determining

bacterial motility, and in promoting anaerobic growth.Solid media (1.5-2% nutrient agar), such as nutrient agar, are used 1) for the surface growth

of microorganisms in order toobserve colony morphology, 2) for pure culture isolation, 3) often in

the enumeration and isolation ofbacteria 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


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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 Petridish. 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 categorized based on their application:

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. (Eg: Mac Conkey agar)

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. (Eg: Mac Conkey agar)

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.

NON-SELECTIVE MEDIA:

Nutrient agar media:

It is the basic bacteriological media used for the isolation of total bacteria. The major components of

media are: Peptone, meat extract, glucose, NaCl and agar.

Potato dextrose agar:

PDA is used for the isolation of total fungi. Major components of the medium are potato, glucose,

agar and acidic pH

Oat meal agar medium:

OMA is used for the isolation of fungi. Commercially available OMA are pre-mixed. These aredehydrated medium. 3.5g of oat meal agar is added and sterilized.

SELECTIVE MEDIA:

Some media have compounds that favour the growth and/or detection of specific microorganisms and

are inhibitory to others.


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Mac conkey agar:

It is the differential plating medium used for the selection and recovery of Enterobacteriaceae and

related Enteric G-ve rods. It contains bile salts, neutral pH, lactose, neutralized crystal violet which

helps to inhibit the growth of G+ve bacteria and this medium helps to differentiate coli from bacilli,

typhi and paratyphi. Coli form bacteria forms acid as a result of lactose fermentation. When this

occurs, the medium surrounding the growth of the bacteria also becomes red because of the action of

the acid that precipitates the bile salts and is followed by absorption of neutral red. Typhoid and

Paratyphoid bacteria are non-lactose fermenters and therefore do not produce acid. Hence, colonies

are colorless or transparent.

DIFFERNTIAL MEDIA:

Differential medium contain substances that permit the detection of microorganisms with specific

metabolic activity. This can distinguish morphologically and biochemically related groups of

organisms. They contain chemicals and other compounds that produce a characteristic change in the

appearance of bacterial growth and of surrounding areas following inoculation and incubation.

Mannitol salt agar:

This medium inhibits the growth of all other bacteria except the Staphylococcus. It contains Mannitol

as Carbon souce which some Staphylococci are capable of fermenting. This also has a pH indicator

for detecting acid produced by mannitol fermenting Staphylococci which exhibits a yellow zone

surrounding their growth and non-fermenters will not produce the colour change.

MATERIALS REQUIRED

Nutrient agar

Nutrient broth

Distilled water

Water bath

2 L Erlenmeyer flask

Autoclave


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COMPOSITION OF NUTRIENT BROTH:

Peptone - 0.5 g

Yeast extract - 0.3 g

NaCl - 0.5 g

Distilled water 100 ml

pH -7 0.2

COMPOSITION OF NUTRIENT AGAR:

Peptone - 0.5 g

Yeast extract - 0.3 g

NaCl - 0.5 g

Distilled water 100 ml

Agar - 2 g

pH -7 0.2

PROCEDURE:

1. Dissolve the ingredients in 100 ml of distilled water.

2. Determine the pH and adjust with 0.1N HCl or 0.1N NaOH if necessary.

3. Take a conical flask with one-third free space and close the mouth with a cotton plug. Cover the

cotton plug with a stuff paper using a rubber band.

4. For solid medium, add the agar and boil to dissolve it.

5. Sterilize the medium by autoclaving at 15Lb pressure for 20 mins.


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RESULT:


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EXPERIMENT NO: 4

CULTURING OF MICROORGANISMSIN

BROTH AND IN PLATES (SPREAD PLATE, POUR

PLATE, STREAK PLATE)


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EXP. NO: 4 CULTURING OF MICROORGANISMS - IN BROTH AND IN PLATES

DATE: (SPREAD PLATE, POUR PLATE, STREAK PLATE)

AIM:

To learn the culturing of microorganism, isolation and preservation of bacterial culture.

PRINCIPLE:

Organisms grown in broth cultures cause turbidity, or cloudiness, in the broth. On agar,

masses of cells, known as colonies, appear after a period of incubation. Certain techniques will allow

bacterial cells to be widely separated on agar so that as the cell divides and produces a visible mass

(colony), the colony will be isolated from other colonies. Since the colony came from a single

bacterial cell, all cells in the colony should be the same species. Isolated colonies are assumed to bepure cultures. Colony morphology is described in terms of shape, margin or edge, elevation and color

(Fig. 1).

Figure 1: Bacterial colony morphology description


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MATERIALS REQUIRED:

Bunsen burner

Inoculatingloopand needle

Glasswaremarkingpencil

Culture media

INOCULATION AND OTHER ASEPTIC PROCEDURES

Essential points

There are several essential precautions that must be taken during inoculation procedures to

control the opportunities for the contamination of cultures, people or the environment.

Operations must not be started until all requirements are within immediate reach and must

be completed as quickly as possible.

Vessels must be open for the minimum amount of time possible and while they are open

all work must be done close to the Bunsen burner flame where air currents are drawn

upwards.

On being opened, the neck of a test tube or bottle must be immediately warmed by

flaming so that any air movement is outwards and the vessel held as near as possible to the

horizontal.

During manipulations involving a Petri dish, exposure of the sterile inner surfaces to

contamination from the air must be limited to the absolute minimum. The parts of sterile pipettes that will be put into cultures or sterile vessels must not be

touched or allowed to come in contact with other non-sterile surfaces, e.g. clothing, the

surface of the working area, outside of test tubes/bottles.

Using a wire loop

Wire loops are sterilized using red heat in a Bunsen flame before and after use. They must

be heated to red hot to make sure that any contaminating bacterial spores are destroyed. The handle

of the wire loop is held close to the top, as you would a pen, at an angle that is almost vertical. This

leaves the little finger free to take hold of the cotton wool plug/ screw cap of a test tube/bottle.

Flaming procedure

The flaming procedure is designed to heat the end of the loop gradually because after use it will

contain culture, which may splutter on rapid heating with the possibility of releasing small particlesof

culture and aerosol formation.


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1. Position the handle end of the wire in the light blue cone of the flame. This is the cool

area of the flame.

2. Draw the rest of the wire upwards slowly up into the hottest region of the flame,

(immediately above the light blue cone).

3. Hold there until it is red hot.

4. Ensure the full length of the wire receives adequate heating.

5. Allow to cool then use immediately.

6. Do not put the loop down or wave it around.

7. Re-sterilize the loop immediately after use.

Using a pipette

Sterile graduated or dropping (Pasteur) pipettes are used to transfer cultures, sterile media

and sterile solutions.

1. Remove the pipette from its container/ wrapper by the end that contains a cotton wool plug,

taking care to touch no more than the amount necessary to take a firm hold.

2. Fit the teat.

3. Hold the pipette barrel as you would a pen but do not grasp the teat.

4. The little finger is left free to take hold of the cotton wool plug/lid of a test tube/bottle and

the thumb to control the teat.

5. Depress the teat cautiously and take up an amount of fluid that is adequate for the amount

required but does not reach and wet the cotton wool plug.

6. Return any excess gently if a measured volume is required.

7. The pipette tip must remain beneath the liquid surface while taking up liquid to avoid the

introduction of airbubbles which may cause spitting and, consequently, aerosol formation

when liquid is expelled.

8. Immediately put the now contaminated pipette into a nearby discard pot of disinfectant.

9. The teat must not be removed until the pipette is within the discard pot otherwise drops of

culture will contaminate the working surface.


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Figure 2: Flaming a loop

Flaming the neck of bottles and test tubes

1. Loosen the lid of the bottle so that it can be removed easily.

2. Lift the bottle/test tube with the left hand.

3. Remove the lid of the bottle/cotton wool plug with the little finger of the right hand (Turn

the bottle, not the lid).

4. Do not put down the lid/cotton wool plug.

5.

Flame the neck of the bottle/test tube by passing the neck forwards and back through a hot

Bunsen flame.

6. Replace the lid on the bottle/cotton wool plug using the little finger (Turn the bottle, not the

lid).


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Figure 3: Flaming the neck of the bottles.

Culturing bacteria in broth

A broth culture is a bacterial culture in which desired bacteria are suspended in liquid broth; a

nutrient medium. The liquid broth is inoculated with the bacteria and left overnight for the bacteria to

grow. It is also referred to as liquid culture. The only difference between broth and agar media is that

broths do not contain an agar component. We use broth tubes primarily for specific assays, or (rarely)

for bacteria that will not form colonies on a solid surface. In broth a species may display motility

and/or a characteristic pattern of association among individual cells, such as chains or clusters, which

is not as obvious in agar cultures.

To prepare broth a dry medium is layered onto the surface of a measured volume of water as

with agar media, mixed, and distributed into individual loosely capped or vented capped tubes in

racks. Heating to dissolve components is sometimes required, but not always. Racks are steam

sterilized and then allowed to cool, and caps tightened to preventevaporation. Unlike preparation of

agar plates, tubes are prepared with media already in the incubation vessel. A large volume syringe

can facilitate distribution of media into individual tubes.

PROCEDURE

1. Light the Bunsen burner.

2. Place the culture you wish to transfer (tube A) near the tube of sterile broth that you will

inoculate (tube B).


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3. Label the tube B with the name of the microorganism and the date

4. Hold the inoculating loop with your thumb and first two fingers. Heat the inoculating loop in

the Bunsen burner until it is red hot. Heat several inches of loop since that much of it will

contact the inside of the tubes. Allow the loop to cool for a few seconds while you hold it in

your hand. Do not put it down or allow the loop to touch any surface after it is sterile.

5. While continuing to hold the inoculating loop with your thumb and first two fingers, pick up

the tube A in your left hand and remove the cap with your thump and first two fingers, pick

up tube A in your left hand and remove the cap with the last two fingers of your right hand.

Keep the cap in your right hand and do not allow it to touch any surface.

6. Using your left hand, draw the open top of tube A gently through the flame of the Bunsen

burner. Do not hold it in the flame for more than a second.

7.

Place the sterile loop out of the tube and continue to hold it in your hand.

8. Draw the open top of tube A gently through the flame of the Bunsen burner and then replace

the cap on the culture tube A. Replace tube A in its rack.

9. While still holding the loop with inoculums in your right hand, pick up the sterile tube of

broth (tube B) with your left hand. Remove the cap with the last two fingers of your right

hand. Keep the cap in your right hand and do not allow it to touch any surface.

10.Draw the open top of the sterile tube B gently through the flame. Do not hold it in the flame

for more than a second.

11.Place the loop containing the droplet of culture into the tube B and gently swirl it to transfer

the microorganisms to the sterile broth.

12.Remove the loop from the broth and continue to hold it in your hand.

13.Draw the open top of the sterile tube B gently through the flame and then replace the cap,

which should still be in your right hand, on tube B. Place tube B back in the test tube rack.

14.Heat the inoculating loop in the Bunsen burner until it is red hot. You can now place it on the

bench or in a rack.

15.

At no time should the inoculating loop, the top of the tubes or the inside of the cap have

touched any non-sterile surface (especially your fingers or hand).


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Culturing bacteria in plate

Spread Plate

The spread plate technique is an easy and direct way of attaining pure culture.When a mixture of

cells is spread on an agar surface, every cell grows into a complete separate colony. A

microscopically visible growth or cluster of microbes on a solid medium can be visualized. Each

colony represents a pure culture. A small volume of dilute microbial mixture containing around 30 to

300 cells is transferred to the centre of an agar plate and spread evenly over the surface with a sterile

bent glass rod. Spread plates can be used to count the microbial population.

Procedure:

1. Pipette 0.1 ml of the sample on the centre of the plate containing agar medium.2. Dip the L-rod into a beaker of ethanol and show it to a flame and allow it to cool.

3. Spread the cells evenly over the solid agar surface with the sterilized L-rod.

4. Incubate the plates at the desired temperature for overnight.

5. Note the results the next day morning.

Streak plate

The loop is used for preparing a streak plate. This involves the progressive dilution of an

inoculum of bacteria or yeast over the surface of solidified agar medium in a Petri dish in such a way

that colonies grow well separated from each other.

The aim of the procedure is to obtain single isolated pure colonies.

1. Loosen the top of the bottle containing the inoculum.

2. Hold the loop in the right hand.

3. Flame the loop and allow to cool.

4. Lift the bottle/test tube containing the inoculum with the left hand.

5.

Remove the lid/cotton wool plug of the bottle/test tube with the little finger of the left hand.

6. Flame the neck of the bottle/test tube.

7. Insert the loop into the culture broth and withdraw.

At all times, hold the loop as still as possible.

8. Flame neck of the bottle/test tube.

9. Replace the lid/cotton wool plug on the bottle/test tube using the little finger. Place


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bottle/test tube on bench.

10.Partially lift the lid of the Petri dish containing the solid medium.

11.Hold the charged loop parallel with the surface of the agar; smear the inoculum backwards

and forwards across a small area of the medium (see figure4 streaked area =A).

12.Remove the loop and close the Petri dish.

13.Flame the loop and allow it to cool. Turn the dish through 90 anticlockwise.

14.With the cooled loop streak the plate from area A across the surface of the agar in three

parallel lines (to B see figure 4). Make sure that a small amount of culture is carried over.


16.Flame the loop and allow to cool. Turn the dish through 90 anticlockwise again and streak

from B across

17.

The surface of the agar in three parallel lines (to C see figure 3).


19.Flame the loop and allow to cool. Turn the dish through 90 anticlockwise and streak loop

across the surface of the agar from C into the centre of the plate (to D see figure 3).

20.Remove the loop and close the Petri dish. Flame the loop.

21.Seal and incubate the plate in an inverted position.


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Figure 4: Streaking of Microorganisms.

Inoculating the cooled molten nutrient agar bottle

1.Pick up the cooled molten nutrient agar bottle.

2.Remove the lid from the molten nutrient agar bottle with the little finger of the right hand

which still holds the charged pipette. Do not put down the lid.

3.Flame the neck of the bottle.

4.Insert the pipette into the bottle and gently release the required number of drops of

inoculum onto the agar.

5.Flame the neck of the bottle and replace the lid.6.Put the pipette into a discard pot. Remove the teat while the pipette is pointing into the

disinfectant.

Pour plate

A pour plate is one in which a small amount of inoculum from broth culture is added by

pipette to a molten, cooled agar medium in a test tube or bottle, distributed evenly throughout the

medium, thoroughly mixed and then poured into a Petri dish to solidify. Pour plates allow micro-

organisms to grow both on the surface and within the medium.

Most of the colonies grow within the medium and are small in size; the few that grow on

the surface are of the same size and appearance as those on a streak plate.

If the dilution and volume of the inoculum, usually 1 cm, are known, the viable count of

the sample i.e. the number of bacteria or clumps of bacteria, per cm can be determined.

Inoculation using a Pasteur pipette


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1. Loosen the top of the bottle containing the inoculum.

2. Remove the sterile Pasteur pipette from its container, attach the bulb and hold in the right

hand.

3. Lift the bottle/test tube containing the inoculum with the left hand.

4. Remove the lid/cotton wool plug with the little finger of the right hand.

5. Flame the bottle/test tube neck.

6. Squeeze the teat bulb of the pipette very slightly, put the pipette into the bottle/test tube and

draw up a little of the culture. Do not squeeze the teat bulb of the pipette after it is in the

broth as this could cause bubbles and possible aerosols.

7. Remove the pipette and flame the neck of the bottle/test tube again, before replacing the

lid/cotton wool plug.

8. Place bottle/test tube on bench.

At all times hold the pipette as still as possible.

Pouring the pour plate

1. Roll the bottle gently between the hands to mix the culture and the medium thoroughly.

Avoid making air bubbles.

2. Hold the bottle in the left hand; remove the lid with the little finger of the right hand.

3. Flame the neck of the bottle.

4. Lift the lid of the Petri dish slightly with the right hand and pour the mixture into the Petri

dish and replace the lid.

5. Flame the neck of the bottle and replace the lid.

6. Gently rotate the dish to ensure that the medium covers the plate evenly.

7. Allow the plate to solidify.

8. Seal and incubate the plate in an inverted position.

Figure 5: Pouring the inoculated medium.


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INCUBATION

The lid and base of an agar plate should be taped together with 2-4 shortstrips of adhesive

tape as a protection from accidental (or unauthorized!) opening during incubation. (Although tape

is the preferred method Parafilm could be used as an alternative for sealing the plates.)

Agar plates must be incubated with the medium-containing half (base) of the Petri dish

uppermost otherwise condensation will occur on the lid and drip onto the culture. This might cause

colonies to spread into each other and risk the spillage of the contaminated liquid.

The advantages of incubators are that they may be set at a range of temperatures and reduce

the possibility of cultures being interfered with or accidentally discarded. However, many cultures

suitable for use in schools will grow at room temperature in the interval between lessons and can

beincubated satisfactorily in a cupboard. The temperature of an incubator varies from the set

temperature, oscillating by several degrees in the course of use.

Water baths are used when accurately controlled temperatures are required, e.g. for enzyme

reactions and growth-temperature relationships, when temperature control of incubators is not

sufficiently precise. They should be used with distilled or deionised water to prevent corrosion and

emptied and dried for storage.

Maintaining stock cultures

It may be convenient to maintain a stock of a pure culture instead of re-purchasing it when

needed. Most of those considered suitable for use are also relatively easy to maintain by sub-

culturing on the medium appropriate for growth but maintenance of stock cultures needs to be well

organized with attention to detail. Be prepared to transfer cultures four times a year to maintain

viability. Cultures on streak plates are not suitable as stock cultures.

Slope/ Slant cultures in screw cap bottles are preferred because the screw cap reduces

evaporation and drying out and cannot be accidentally knocked off (cf. a streak plate culture).

Slope cultures are preferred to broth (i.e. liquid medium) cultures because the first sign of

contamination is much more readily noticed on an agar surface. Two stock cultures should be

prepared; one is the working stock for taking sub-cultures for classes, the other is thepermanent stock which is opened only once forpreparing the next two stock cultures. Incubate at

an appropriate temperature until there is good growth.

For growing strict aerobes it may be necessary to slightly loosen the cap for incubation (but

close securely before storage) if there is insufficient air in the headspace.

As soon as there is adequate growth, store the cultures at room temperature in either a


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cupboard or drawer. Keep on the lookout for contamination.

Checking cultures for contamination

Evidence for a culture being pure or otherwise is given by the appearance of colonies on a

streak plates and of cells in a stained microscopical preparation. There should be uniformity of

colony form and cell form (and consistency with the appearance of the original culture!). It is

sensible to check purity on suspicion of contamination of the working stock culture from time to

time and of the permanent stock when preparing new stock cultures.

If a culture becomes contaminated, it is not advisable to try to remedy the situation by

taking an inoculum from a single colony from a streak plate of the mixed culture because of the

possibility of(1) not being able to distinguish between the colony forms of the contaminant and the

original culture, and (2) culturing a variant of the original culture that does not behave as the

original culture did. Instead, go back to the working (or permanent) stock cultures; tha t are what

they are for! Preventing contamination of cultures and the environment.

RESULT:


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VIVA QUESTIONS:

1. How will you avoid contamination during culturing the microorganisms?

2. What is streak plate method and pour plate method?

3. What is Swabbing?

4. What are the different types of streaking methods available?

5. How will you prevent the contamination of culture?


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EXPERIMENT NO: 5

STAINING TECHNIQUES & MOTILITY

TEST


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EXP. NO: 5a STAINING TECHNIQUESGRAM STAINING

DATE:

AIM:

To perform Gram stain to differentiate two principal groups of bacteria: Gram +ve and

Gram -ve

PRINCIPLE:

There are several types of stains which are commonly used in microbiology. The first is a

simple stain, which uses only one reagent which provides contrast between the background and

the heat-fixed bacterium itself. The bacterium takes up stain and becomes colored, while the

background remains unstained. Simple stains are typically used on bacterial smears which have

been heat-fixed and thus contain non-living microbes.

A second type of stain is a negative stain, which uses a single reagent to provide contrast

between the background and the living bacterium. Thus, the background is stained, while

bacterium does not take up any stain. Negative stains are typically used when observing live

bacteria is desired.

A differential stain is a type of staining that allows you to distinguish between types of

bacteria or between specific structures in a bacterium for example, Gram Positive and Gram Negative

bacteria. A differential stain typically uses two or more reagentsa primary stain (Crystal Violet)

and a counter stain (Safranin). Bacteria stain differentially because of chemical and physical

differences in their cell wall. G +ve cell wall consists of many layers of Peptidoglycan. The

Crystal Violet iodine complex is larger than than the crystal violet or iodine molecules that

initially enter the cell and hence it cannot pass through the thick peptidoglycan layer. So the

counter stain cannot be absorbed by the cells and it retains the primary stain colour. In Gram ve

cells, alcohol dissolves the outer lipopolysaccharide layer and the Crystal violet iodine complex

will be washed out through the thin layer of peptidoglycan. So, Gve cells will absorb the counter

stain.

Chemically, there are two main types of stains: basic stains, which have a positivecharge(cationic) and acidic stains, which have a negative charge (anionic). Basic stains have an affinity

for negative components of cells, and include dyes such as methylene blue, crystal violet, and

carbolfuchsin. Acidic stains have an affinity for positive components of cells, and include dyes

such as nigrosin, India ink, and picric acid. Since cell walls are negatively charged, a positive dye

will be attracted to and stain the cell wall, whereas a negative dye will be repulsed by the cell wall


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and not directly stain the cell.

PROCEDURE FOR PREPARING A BACTERIAL SMEAR

1. Obtain a glass slide and clean if necessary.

2. Using a broth culture:

a.

Gently agitate your culture broth tube to disperse the bacteria.

b. Sterilize your inoculating loop using an incinerator or a Bunsen burner, and

let cool for 20-30 seconds.

c. Place loop in the bacterial broth and put the loopful of the broth onto the glass

slide. Rub the drop into a nickel-sized smear. Sterilize the loop again to kill any

remaining bacteria. Let the smear air dry completely. Do not use heat to dry

your smear!

3. Using an agar plate:

a. Place a smalldrop of water in the center of the slide.

b. Sterilize your inoculating loop using an incinerator or a Bunsen burner, and let cool

for 20-30 seconds.

c. Use the sterile loop to pick up a small amount of bacterial growth from the surface

of the plate. Do not dig into the agar. Put the loopful of bacteria into the drop of

water on the glass slide, and rub the drop into a nickel-sized smear. Sterilize the

loop again to kill any remaining bacteria Let the smear airdry completely. Do not

use heat to dry your smear!

4. Heat fixthe slide.

a. Incinerator method: Hold the slide with a wooden clothes pin approximately

1cm over the barrel of a hot incinerator for 2030 seconds. Let the slide cool.

b. Bunsen burner method: Hold the slide with a wooden clothes pin, and pass 10-

12 times through the flame. Let the slide cool.

5. Optional: After the slide has cooled us a marker or wax pencil to outline the area of

thesmear on the underside of the slide. This will help you locate your sample later.

GRAM STAIN

The Gram stain is a differential stain which distinguishes bacteria based on cell wall

properties. Bacterial cell walls are composed primarily of peptidoglycan and bacteria can be

classified into two main groups dependent on the amount of peptidoglycan present in their cell


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wall. Gram-positiveorganisms have a thick layer of peptidoglycan, whereas Gram-negative

organisms have a thin layer of peptidoglycan, plus an additional outer membranethat is absent

in Gram-positive organisms.

In the gram staining procedure, the primary stainis crystal violet, and all cells take up

the purple crystal violet stain. Following the primary stain, Grams Iodine is applied to

thebacterial smears. The iodine acts as a mordant,enhancing the ability of the stain to enter

and bind to the bacteria. Specifically, the iodine binds with crystal violet and locks it into

peptidoglycan of bacteria. It also intensifies the purple color. The decolorizing agentused in

the gram staining procedure is 95% ethanol, which is a lipid solvent that melts the Gram-

negative outer membrane and leads to decolorization of Gram-negative cells. It also dehydrates

proteins, helping the primary stain to remain in Gram-positive cell walls. The counter stain

then used is Safranin, which stains the decolorized Gram-negative cells pink. Thus, at the endof

the staining procedure, Gram-positive cells are purple and Gram-negative cells are pink.

Note: It is preferable to use fresh cultures for the Gram stain. Old cultures may stain Gram-

variable (a mix of purple and pink) because they decolorize easily.

CULTURES NEEDED:

Nutrient broth tubes or plates of the following:

Escherichia coli

Staphylococcus xylosus

Bacillus megaterium

PROCEDURE:

1. Prepare a bacterial smear with a mixture of all 3 organisms (i.e. 1-2 loop full of each) listed

above and heat fixit.

2. Place the slide on a staining tray, and cover the smear with crystal violet. Allow to stain for

60 seconds.

3. Tilt the slide and gently rinse with distilled water until the stain is removed.


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4. Cover the smear with Grams Iodine, and allow to sit for 60 seconds.

5. Tilt the slide and gently rinse with distilled water.

6. IMPORTANT STEP: Tilt the slide and let 2-3 drops of Decolorizer run over the slide.

If the last drop is still purple, continue decolorizing, 2-3 drops at a time, until the

decolorizer runs clear. Rinse with distilled water.

7. Cover the smear with Safranin, and stain for 45 seconds.

8. Tilt the slide and rinse with distilled water.

9. Place the slide in a book of Bibulous paper and blot to dry. You do not need a cover slip!

Observe the slide under oil immersion, and draw what you see in the results section below.

You should see: Small purple cocci (spheres) which are the gram-positive S.xylosus, large

purple rods, which are the gram-positiveB. megaterium, and small pinkrods, which are the

gram-negativeE. coli. Labelthese in your drawing.

10.

Clean your microscope with lens cleaner, paying extra attention to the 40X and 100X

objectives.

OBSERVATION:


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RESULT:


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VIVA QUESTIONS:

1. What are the several advantages of differential staining procedures compared with simplestaining techniques.

2. Give the purpose of each of the following reagents in a differential staining procedure:

a. Primary stain

b. Counter stain

c. Decolorizing agent

d. Mordant.


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3. Why is it important for the counter stain to be a lighter color than the primary stain?

4. What is the purpose of staining bacteria?

5. What is the most common differential staining procedure used in microbiology?


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EXP. NO: 5b MOTILITY TEST

DATE:

AIM:

To learn to make hanging drop slides and then use this technique to observe the motility of

the live bacteria

PRINCIPLE:

Motility is an important characteristic used to identify microorganisms. Three bacteria will

be used which vary in size, shape and arrangement of flagella, and types of motion. Specifically

Pseudomonas aeruginosa, a monotrichous bacterium exhibits high motility using a polar

flagellum, Bacillus cereusthat moves by peritrichous flagella and Spirilliumvolutans, a helical cell

that moves using large bipolar tufts of flagella (possible mixture of amphitrichous and

lophotrichous organisms) (Refer to Figure 1).Flagellae are, in effect, rotary motors comprised of a number of protein rings embedded in

the cell wall. In action the filament rotates at speeds from 200 to more than 1,000 revolutions per

second, driving the rotation of the flagellum. The direction of rotation determines the movement

of the cell. Counterclockwise rotation of polar flagella thrusts the cell forward with the flagellum

trailing behind. Periodically, the direction of rotation is briefly reversed, causing what is known as

a "tumble" resulting in reorientation of the cell.


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Investigation of the movement of live bacteria by microscope is possible e. g. with hanging-

drop preparation (Figure 2). A suspension of microorganisms is placed in the centre of a cover slip

and turned over with a special glass slide with a hollow depression in the centre. When observing live

bacteria, be careful not to confuse motility with Brownian motion resulting from bombardment by

water molecules.

In Brownian motion, organisms all vibrate at about the same rate and maintain a relatively

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.

Figure 2:Hanging drop preparation: (a) From the examined bacterial culture, (b) Prepare a

weak suspension in a drop of water in the center of cover slip. (c) Put the glass slide with the

hollow depression upside down over the cover slip preparation so that the drop of the culture is

in the center of the depression, and then quickly turn it over.

MATERIALS ANDEQUIPMENTS:

Glass slide with hollow depression

Cover slip

Inoculating loop

Bunsen burner

Pipette, sterile pipette tips

Light microscopy

Immersion oil

Benzene


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PROCEDURE:

1. Clean the cover slip such that it is free from grease and wipe it with a dry cotton tissue.

2. Place a thin film of mountant around the edge of the cover slip.

3. Place a loop full of culturein the centre of the cover slip.

4. Turn the slide carefully upside down to make the drop hang in the cavity.5. Observe the edge of the dropusing a high power objective under a microscope to visualize the type

of motility of the bacteria.

OSERVATION:


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RESULT:


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VIVA QUESTIONS:

1. Name some organisms that are motile in nature.

2. What is the importance of performing motility test?

3. Explain the term monotrichous

4. What is chemotaxis?

5. What is gliding motility?


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EXPERIMENT NO: 6

QUANTITATION OFMICROORGANISMS


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EXP. NO: 6 QUANTITATION OFMICROORGANISMS

DATE:

AIM:

To learn the technique that will help in quantifying the number of microorganisms present in

the given sample.

PRINCIPLE:

The counting of bacteria is important if you want to know the number of bacteria in a sample.

The common methods are: Plate count, Direct count, and Turbidometric.

Plate count- is used on the premise that each viable bacterium will produce a colony when

growing on a agar plate. A sample of the material to be counted is suspended in liquid and placed in

a empty petri plate. Next, melted agar is poured into the plate. After incubation, each organism

produces a colony in the agar that can be counted.

There are two main advantages of the plate count over other methods. Only viable organisms

are counted, which are the ones considered to be important. Samples with small methods can also be

counted. The disadvantages are related to size and frequency. Bacteria are usually present in large

numbers. E.coli could easily contain over one billion cells/ml. Some bacteria will stick together,

giving rise that two different organisms may produce the appearance of only one colony.

Direct count - Organisms in a suspension of bacteria are placed on a slide that has been ruled

into squares and can hold a specific amount of volume. By counting the bacteria that appear on the

grid areas, the number of organisms in a sample can be calculated.

The direct count is much faster than the plate count but has its disadvantages. There must

be a certain multiple number of organisms before there are enough to be seen, and both viable

and nonviable organisms appear the same under the microscope.

Turbidometric - turbid simply means cloudy. In this method, a spectrophotometer measures

the turbidity on bacteria in a broth. The more bacteria present, the cloudier the broth.

PLATE COUNT: PROCEDURE

The purpose of this exercise is to quantify the number of bacteria in a broth culture of E.

coli. In microbiological research, it is often necessary to be able to quantify the number of

living bacteria in a particular sample. One of the major ways to do this is using viable plate

counts, in which bacterial cells from a liquid culture are spread onto an agar plate. The plate is


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incubated, the number of colonies that grow on the plate are counted, and the number of original

bacterial cells in the culture is determined. In most cases, however, the liquid culture being

quantified contains too many cells to be directly plated onto agar plates there would be so

much growth that it would be impossible to count individual colonies! Therefore, the liquid

culture needs to be diluted, often 1-million-fold, before it can be plated.

When such a large dilution is required, an accurate dilution cannot be made in a single

dilution step and it is necessary to make serial dilutions. Serial dilutions are a step-wise set of

dilutions which sequentially dilute the bacterial culture. One or more of the dilutions are then plated

on the agar plates to determine the number of colonies present in the original culture.

Figure 1: Serial Dilution.

Only plates containing between 30 and 300 colonies are counted to ensure statistically

significant data. To estimate the number of bacterial in the original culture, the # of colonies on the

plate is multiplied by the total dilution plated. For example, suppose 0.1 ml of a 10-6dilution was

plated, and 123 colonies were counted following incubation. The total dilution platedwould be 10-

7(since only 0.1 ml was plated), and the number of bacteria/ml of the original culture would be: (123) x

1/10-7= 1.23 x 109CFU/ml. Note that the results are expressed as colony forming units(CFU) per ml.

MEDIA NEEDED: (per group of four)

3 Nutrient agar plates

7 Dilution blanks containing 9 ml water


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CULTURES NEEDED:

Overnight broth culture ofEscherichia coli

PROCEDURE:

1. Label the dilution blanks as follows: 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7.

2.

Label the agar plates as follows: 10-6, 10-7and 10-8.

3. Using a sterile pipette, transfer 1 ml of the E. colibroth culture into the tube labeled 10-1. Mix

thoroughly.

4. Using a new sterile pipette, transfer 1 ml of the 10-1 tube into the tube labeled 10-2. Mix

thoroughly.


thoroughly.

6. Using a new sterile pipette, transfer 1 ml of the 10 -3tube into the tube labeled 10-4. Mix

thoroughly.


thoroughly.

8. Using a new sterile pipette, transfer 1 ml of the 10 -5tube into the tube labeled 10-6. Mix

thoroughly.


thoroughly.

10.Using a new sterile pipette, transfer 0.1 ml of the 10-5tube to the nutrient agar plate labeled 10-6,

and spread the liquid thoroughly and evenly o

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