practice physiology of microorganisms

8/4/2019 Practice Physiology of Microorganisms

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Physiology of

microorganisms


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Classification of mediaby practical application

simple media (nutrient broth, nutrientagar);

enriched media (sugar agar, serum agar,blood agar, etc.);

selective media (alkaline peptone water,

egg yolk salt agar, bile broth, etc.); differential media (Endos medium,

Levinas medium, Gisss medium, etc.).


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Growth of microorganismon nutrient agar


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Staphylococci on blood agar


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Pneumococci (Streptococcuspneumoniae) on blood agar


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Classification of hemolysison blood agar


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Selective mediumEgg yolk salt agar

negative result (lecithinase-) positive result (lecithinase+)
http://en.wikipedia.org/wiki/File:Clostridium_perfringens_01.png


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Selective mediumV.choleraeon TCBS Agar

V.choleraetolerate alkaline media (pH>8.2) that kill most

intestinal commensals, but they are sensitive to acid.


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Selective mediumLowenstein-Jensen medium used for

growing Mycobacterium tuberculosis


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Selective medium for C.diphtheriae


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Selective medium:aculture of Legionellaon Selective Buffered

Charcoal Yeast Extract agar


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Differential medium: Endo agarlac- lac+


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Salmonellaon Endo agarlac- colonies


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E.colion Endo agar (lac+ colonies)


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Differential medium:E.colion Levine agar

(Eosin-Methylene Blue (EMB) agar):lac+ colonies


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Differential medium: MacConkey agar

lac+ colonies lac- colonies


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Differential medium:Gisss mediumnegative result, positive result without gas, positive result with gas


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Drigalskys methodThe process of isolation and identification of pure

culture of microorganism belonging to aerobes orfacultative anaerobes consists of three stages: I stage. Inoculation of agar plate: The streak plate is

used primarily for isolating microorganisms in pureculture from specimens containing a mixed flora.

Incubation. II stage. Obtaining isolated colonies on platespermits a study of cultural characteristics. Each typeof isolated colony should be stained for studyingcellular morphology (Gram method etc.) andinoculated on solid agar slant. Incubation.

III stage. Identification of isolated pure culture ismade by examining morphology of microorganismsand studying their morphological, staining, cultural,biochemical, antigenic and virulent properties andsusceptibility to phages, chemical substrates,antibiotics etc.


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I stage of Drigalsky method
http://2.bp.blogspot.com/_TCTZwIba15E/SqxSxaeGi3I/AAAAAAAAAII/0fJfQc2nim8/s1600-h/streak1.gifhttp://2.bp.blogspot.com/_TCTZwIba15E/SqxNw3XsEjI/AAAAAAAAAFY/AIj7l-_3dOg/s1600-h/00labtechimagexii.jpg


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II stage of Drigalsky method

Cultural characteristics of bacteria are described as follows: Shape: Circular, irregular, radiate or rhizoid. Surfaces: Smooth, rough, fine or coarsely granular, papillate, glistening,

etc. Size: Surface of colony is measured in millimeter. Elevation: Raised, low convex dome, umblicate. Some bacteria produce

spreading growth (for example, Proteus). Edges: Entire, crenated, fimbriated or effuse. Colour: Some microorganisms may produce pigmented colonies. Opacity: Colonies on nutrient agar may be transparent, translucent or

opaque. Consistency: Colonies may be hard or firm, friable and membranous, soft

and butyrous. Changes in the medium: Some microorganisms produce beta type of

hemolysis around the colony on blood agar. Few bacteria producesoluble pigment that diffuses into the medium.

Emulsifiability: easily or not.


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II stage of Drigalsky method


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Examples of various colony morphologies. The appearance of colonieson a plate is species specific and can be very helpful in identifying

isolates.


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Examples of various colony morphologies. The appearance of colonieson a plate is species specific and can be very helpful in identifying

isolates.


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Colony of Staphylococcus(you can describe shape, surface, elevation, edges, color, opacity even by this

photo without any problem)


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Some bacteria produce spreading growth(for example, Proteus).


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Some microorganisms produce beta type of hemolysisaround the colony on blood agar (S.pyogenes).


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Colony of pneumococcus

(S.pneumoniae)obviousbeta type of hemolysis


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Few bacteria produce soluble pigment that diffusesinto the medium (P.aeruginosaon nutrient agar).

Mycobacterium tuberculosis colonies on a


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Mycobacterium tuberculosiscolonies on aplate - note rough colonies

(generation time for M.tuberculosisis 15 h,thus, the colonies can appear in 2-8 weeks of cultivation)


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III stage of Drigalsky method:

biochemical identification- indole test +


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biochemical identification- urease test +


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biochemical identificationVoges-Proskauer test


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biochemical identification- Citrate utilization test +

DETERMINATION OF WAYS OF UTILIZATION OF


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DETERMINATION OF WAYS OF UTILIZATION OFGLUCOSE ON OXIDATIVE-FERMENTATIVE MEDIA

(E.coliinside medium with glucoseunder oil (anaerobic utilization positive result) and without oil (aerobic

utilization also positive result)


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Biochemical identification:Kligler agar

The Kligler's iron agar is a test tube thatcontains agar, a pH-sensitive dye (phenol red),1% lactose, 0.1% glucose, as well as sodiumthiosulfate and ferrous sulfate or ferrous

ammonium sulfate. All of these ingredients are mixed together and

allowed to solidify in the test tube at a slantedangle. The slanted shape of this mediumprovides an array of surfaces that are eitherexposed to oxygen-containing air in varyingdegrees (an aerobicenvironment) or notexposed to air (an anaerobicenvironment).
http://en.wikipedia.org/w/index.php?title=Kligler%27s_iron_agar&action=edit&redlink=1http://en.wikipedia.org/wiki/Agarhttp://en.wikipedia.org/wiki/Phenol_redhttp://en.wikipedia.org/wiki/Lactosehttp://en.wikipedia.org/wiki/Glucosehttp://en.wikipedia.org/wiki/Sodium_thiosulfatehttp://en.wikipedia.org/wiki/Sodium_thiosulfatehttp://en.wikipedia.org/wiki/Ferrous_sulfatehttp://en.wikipedia.org/wiki/Ferrous_ammonium_sulfatehttp://en.wikipedia.org/wiki/Ferrous_ammonium_sulfatehttp://en.wikipedia.org/wiki/Ferrous_ammonium_sulfatehttp://en.wikipedia.org/wiki/Ferrous_ammonium_sulfatehttp://en.wikipedia.org/wiki/Ferrous_sulfatehttp://en.wikipedia.org/wiki/Sodium_thiosulfatehttp://en.wikipedia.org/wiki/Sodium_thiosulfatehttp://en.wikipedia.org/wiki/Glucosehttp://en.wikipedia.org/wiki/Lactosehttp://en.wikipedia.org/wiki/Phenol_redhttp://en.wikipedia.org/wiki/Agarhttp://en.wikipedia.org/w/index.php?title=Kligler%27s_iron_agar&action=edit&redlink=1


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Biochemical identification:

Kligler agar

Standard system API-20


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Standard system API-20for biochemical identification of bacteria

culture

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AIdentification

8030 + + + + + + + + + + + + + +Klebsiella

pneumoniae

8068 + + + + + + + + Proteus

vulgaris

8P14 + + + + + + + + +Salmonella

sp.


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Anaerobe jars for anaerobicincubation of strict anaerobes

A bi b f bi


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Anaerobic box for anaerobic

incubation of strict anaerobes

Anaerobic transport medi m


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Anaerobic transport medium

containing reducing agents


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The growth of a bacterial culture can be represented by acurve that consists of four stages or phases:

Lag phase - growth and reproduction are just beginning

Log phase - reproduction is occurring at an exponential rateStationary phase - environmental surroundings and food

supply cannot support any more exponential growthDeath phase - when all of the nutrients have been exhausted,

the population dies off

T l f b i


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Total count of bacteria:changes in turbidity can be measured to assess growth of bacteria in

clear medias / broth. This can be conducted using aspectrophotometer or a tubidometer and provides rapid data. Opticaltechniques however, do not typically differentiate between live anddead cells. The standards of turbidity are presented on the photo.


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Viable count of bacteria

(standard plate count)The SPC (standard plate count) is still the single

most common method of enumerating cells. A precisevolume of liquid (media / sample) containing the cells is

placed on the surface of an agar-containing petri plate.The plate is incubated (typically >24 hours) and the plateis counted for colonies. Each colony is assumed to bederived from a single bacterial cell - allowing themicrobiologist to relate the number of colonies (CFU -

colony forming unit) and dilution factors to determine theoriginal number of organisms in the sample. This isusually expressed as CFU / ml (colony forming units permilliliter).


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Dil i f li i l i


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Dilutions of clinical specimenon Petri dishes

C l l i f l i


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Calculation of colonies(by special lattice)


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Disk diffusion method

Zones of inhibition of growth around the disks are measured


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Zones of inhibition of growth around the disks are measured.Diameter of zone indicates the susceptibility or resistance to each

agent: 30 mm means high susceptibility.


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BrothDilution

test

practice physiology of microorganisms

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