Physiology of microorganisms

Classification of media by practical application

simple media (nutrient broth, nutrient agar); 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.).

Growth of microorganism on nutrient agar

Staphylococci on blood agar

Pneumococci (Streptococcus pneumoniae) on blood agar

Classification of hemolysis on blood agar

Selective medium Egg yolk salt agar

negative result (lecithinase-) positive result (lecithinase+)

Selective medium V.cholerae on TCBS Agar

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

intestinal commensals, but they are sensitive to acid.

Selective medium
Lowenstein-Jensen medium used for growing Mycobacterium tuberculosis

Selective medium for C.diphtheriae

Selective medium:
a culture of Legionella on Selective Buffered Charcoal Yeast Extract agar

Differential medium: Endo agar

laclac+

Salmonella on Endo agar

lac- colonies

E.coli on Endo agar (lac+ colonies)

Differential medium:
E.coli on Levine agar
(Eosin-Methylene Blue (EMB) agar): lac+ colonies

Differential medium: MacConkey agar

lac+ colonies lac- colonies

Differential medium: Gisss medium

negative result, positive result without gas, positive result with gas

Drigalskys method

The process of isolation and identification of pure culture of microorganism belonging to aerobes or facultative anaerobes consists of three stages: I stage. Inoculation of agar plate: The streak plate is used primarily for isolating microorganisms in pure culture from specimens containing a mixed flora. Incubation. II stage. Obtaining isolated colonies on plates permits a study of cultural characteristics. Each type of isolated colony should be stained for studying cellular morphology (Gram method etc.) and inoculated on solid agar slant. Incubation. III stage. Identification of isolated pure culture is made by examining morphology of microorganisms and studying their morphological, staining, cultural, biochemical, antigenic and virulent properties and susceptibility to phages, chemical substrates, antibiotics etc.

I stage of Drigalsky method

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 produce soluble pigment that diffuses into the medium. Emulsifiability: easily or not.

II stage of Drigalsky method

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

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

Colony of Staphylococcus
(you can describe shape, surface, elevation, edges, color, opacity even by this photo without any problem)

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

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

Colony of pneumococcus (S.pneumoniae)

obvious beta type of hemolysis

Few bacteria produce soluble pigment that diffuses into the medium (P.aeruginosa on nutrient agar).

Mycobacterium tuberculosis colonies on a plate - note rough colonies

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

III stage of Drigalsky method: biochemical identification - indole test +

III stage of Drigalsky method: biochemical identification urease test +

III stage of Drigalsky method: biochemical identification Voges-Proskauer test

III stage of Drigalsky method: biochemical identification - Citrate utilization test +

DETERMINATION OF WAYS OF UTILIZATION OF GLUCOSE ON OXIDATIVE-FERMENTATIVE MEDIA

(E.coli inside medium with glucose under oil (anaerobic utilization positive result) and without oil (aerobic utilization also positive result)

Biochemical identification:

Kligler agar
The Kligler's iron agar is a test tube that contains agar, a pH-sensitive dye (phenol red), 1% lactose, 0.1% glucose, as well as sodium thiosulfate and ferrous sulfate or ferrous ammonium sulfate. All of these ingredients are mixed together and allowed to solidify in the test tube at a slanted angle. The slanted shape of this medium provides an array of surfaces that are either exposed to oxygen-containing air in varying degrees (an aerobic environment) or not exposed to air (an anaerobic environment).

Biochemical identification:

Kligler agar

Standard system API-20 for biochemical identification of bacteria

culture no.

O N P G +

A D H

L D C + +

O D C +

C I T +

H 2 S + +

U R E + +

T D A +

I N D +

V P

G E L +

G L U + + +

M A N + +

I N O +

S O R + +

R H A + +

S A C + +

M E L + +

A M Y + +

A R A + +

Identification

8030 8068 8P14

Klebsiella pneumoniae Proteus vulgaris Salmonella sp.

Anaerobe jars for anaerobic incubation of strict anaerobes

Anaerobic box for anaerobic incubation of strict anaerobes

Anaerobic transport medium containing reducing agents

The growth of a bacterial culture can be represented by a curve that consists of four stages or phases: Lag phase - growth and reproduction are just beginning Log phase - reproduction is occurring at an exponential rate Stationary phase - environmental surroundings and food supply cannot support any more exponential growth Death phase - when all of the nutrients have been exhausted, the population dies off

Total count of bacteria:

changes in turbidity can be measured to assess growth of bacteria in clear medias / broth. This can be conducted using a spectrophotometer or a tubidometer and provides rapid data. Optical techniques however, do not typically differentiate between live and dead cells. The standards of turbidity are presented on the photo.

Viable count of bacteria (standard plate count)

The SPC (standard plate count) is still the single most common method of enumerating cells. A precise volume 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 plate is counted for colonies. Each colony is assumed to be derived from a single bacterial cell - allowing the microbiologist to relate the number of colonies (CFU colony forming unit) and dilution factors to determine the original number of organisms in the sample. This is usually expressed as CFU / ml (colony forming units per milliliter).

Dilutions of clinical specimen on Petri dishes

Calculation of colonies
(by special lattice)

Disk diffusion method

Zones of inhibition of growth around the disks are measured. Diameter of zone indicates the susceptibility or resistance to each agent: <15 mm mean weak susceptibility; 15-30 mm means medium susceptibility; >30 mm means high susceptibility.

Broth Dilution test