BACTERIAL IDENTIFICATION

METHODS

CONTENT

Purification of cultures

Morphological and pure culture studies

Biochemical tests

PURIFICATION OF CULTURES

Reason to purify cultures.

To characterize an individual species.

To study the morphology and physiology of individual bacterial species

To study their biochemical behavior and response.

To purify, pure cultures techniques can be used.Method:

Streak plate method

Pour plate method

Spread plate method

IMPORTANT PROCEDURE!!

Need to have a control procedure to avoid contamination.

Specimen collection

Preparation of media

Microbiological tecniques

Staining and reagents

Equipment used.

SPECIMEN COLLECTION

Applied the sterile techniques

Use correct media for transportation and stock.

The transport media used to preserve and ensure the viability of bacteria during the transportation period

Important! Label your specimen.

Crucial for cerebrospinal fluid, blood culture and fecal specimens, etc.

USING STERILE TECHNIQUES

Bacteria are everywhere

Media used for bacteria growth welcoming for many bacteria

We only want specific ones to grow Sterile techniques

Sterile remain sterile as long as doesn’t touch anything that isn’t sterile

Also avoid prolonged exposure to air

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ASEPTIC TECHNIQUE:

These are various techniques that are used to minimize the introduction of microorganisms into media especially during transfer processes, such as :pouring of media into Petri dishesinoculation of cultures

These techniques include: cleaning the bench top work areas with

disinfectant solutionwashing hands before starting workother specific techniques that will be

demonstrated in the lab.

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STERILE TECHNIQUES: WHAT CAN YOU DO IN THE LAB?

Wash your hands Keep your bench clean Wear gloves Flame loop, neck of tube Keep cap facing down Work quickly and efficiently Limit talking when opening

cultures

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PREPARATION OF MEDIA

The media should be packed well to prevent from leakage and breaks, protected from moisture and sunlight and excessive heat

The expiry date should be noted and the instruction of storage should be followed

The mix bacterial colonies should be sub cultured until the culture are purified

the bacterial colony characteristic should only derive from a single colony

CULTURE MEDIA

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Plate

Slant

Broth

Deep

MORPHOLOGICAL AND PURE CULTURE STUDIES

Morphological studies:

- Sizes, shapes, cell arrangement, cell wall, surface adherents or appendages,flagella,pili,endospores,ribosomes.

- Macroscopic examintation

Techniques used in the study:

- Microscopic examintion

- Staining techniques

MORPHOLOGICAL AND PURE CULTURE

STUDIES

ISOLATION OF PURE BACTERIAL CULTURES

Divide into 3 groups:

Selective media

Differental media

Enrichment media

SELECTIVE MEDIA

Prepared by the addition of specific subtances to a culture medium that will permit growth of one bacteria while inhibiting the growth of others.

Contain antimicrobial agents such as crytal violet,bile salts,sodium azide,antibiotic and e.t.c.

Salmonella-Shigella Agar- media contain bile salts (inhibits many coliform bacteria).Produce colorless colonies (unable to ferment lactose)

Mannitol Salt Agar -Isolation of Staphylococci.

Bismuth Sulfite Agar-Isolation for Salmonella typhi.Reduces the sulfite to sulfide results in black colonies and with metallic sheen.

DIFFERENTIAL MEDIA

The incorporation of certain chemicals into a medium may result in diagnostically useful growth or visible change in the medium after incubation.

Eosin Methylene Blue(EMB)- Differentiate between lactse and non-lactose fermenters.

Mac Conkey Agar-contain crystal violet and bile salts.Use for selection of Enterobacteriaceae and related gram negative rods.

Hektoen Enteric Agar-High concenration of bile salts.Inhibit Gram positive bacteria and retards the growth of many coliform strains.

IN MACCONKEY AGAR

IN MACCONKEY AGAR

ENRICHMENT MEDIA

These are routinely employed in a laboratory e.g. nutrient broth, nutrient agar, infusion broth,blood agar.

They support the growth of fastidious bacteria.

IN NUTRIENT AGAR

PURE COLONY

IN BLOOD AGAR

HEMOLYSIS Destruction of erythrocytes nd hemoglobin in

medium.

Can be divided into 3 categories:alpha hemolysis, beta hemolysis and gamma hemolysis

Alpha hemolysis-greenish to brownish discolouration around the colonies. (Streptococous gordonii,Streptococcus pneumoniae)

Beta hemolysis-complete lysis of blood cell resulting in clearing effect around the growth of colony.(S.aureus)

Gamma hemolysis-no change in the medium.(Enterococcus faecalis)

BIOCHEMICAL TESTS

Catalase test

Oxidase test

Coagulase test

Sugar fermentation test

MRVP test

Indole test

Citrate test

Motility test

H2S test

Litmus milk test

CATALASE TEST

Produce bubble just after attaching the bacteria to the reagent

To differentiate staphylococci and streptococci

OXIDASE TEST

Have 2 methods:Filter paper/Sterile swab

To help identify Vibrio, Neisseria, Pasteurella and Pseudomonas sp.

Oxidase enzymes oxydize phenylenediamine.

Deep purple colour on reagent paper

OXIDASE TEST

COAGULASE TEST

To identify S.aureus

The enzyme coagulase clots plasma

Tube : fibrin clot

Slide: clumping of bacterial cells

SUGAR FERMENTATION TEST

Glucose test

Maltose test

Sucrose test

Lactose test

Some will appear with gas production

VOGES-PROSKAUER TEST

To differentiate enterobacteria

Organism ferments glucose with acetoin production. Acetoin is oxidised to diacetyl which reacts with creatine.

Brick red colour develop slowly

Eg: E.coli (-)

Klebsiella sp. (+)

METHYL RED TEST

To differentiate Enterobacteria.

Detect the production of sufficient acid during fermentation of glucose in buffered medium to give a colour change of indicator

Brick red medium

INDOLE TEST

Using Kovac reagent.

To differentiate Gram negative rods, especially E.coli .

Demonstrates the ability of certain bacteria to decompose amino acid tryptophan to indole which accumulates in the medium.

Reddening of strip or medium

INDOLE TEST USING OTHER REAGENT

CITRATE TEST

Test the ability of organism to utilise citrate as a sole carbon source and ammonium salt for nitrogen.Result in alkalinization in the medium with colour change indicator.

Use Koser’s liquid citrate medium.

Differentiate Enterobacteria from other bacteria.

Positive result : Blue and turbid medium

MOTILITY TEST

LITMUS TEST

Medium consisting of LACTOSE,CASEIN and the pH indicator azolitmin.

It is used to differentiate members within the genus Clostridium. It differentiates Enterobacteriaceae from other Gram-negative bacilli based on enterics' ability to reduce litmus.

The skim milk provides nutients for growth. The protein is casein and the lactose is for fermentation.

Azolitmin is purple between pH of 4.6 and 8.2. It turns pink when pH reaches 4.5 and blue at a pH of 8.3.

Because of this, litmus milk can give quite unreliable results .

Thus, you would be advised to use litmus milk as a confirmatory test but not a definitive test (except as a last resort).

TRIPLE SUGAR ION

Triple Sugar Iron medium is a differential medium that can distinguish between a number of Gram-negative enteric bacteria based on their physiological ability (or lack thereof) to:

a. metabolize lactose and/or sucroseb. conduct fermentation to produce acidc. produce gas during fermentationd. generate H2S.

TERMS FOR TODAY

Culture collection centre.

ATCC American type culture Collection Centre

NCTCC National Collection of Type Culture

NCIM Natonal Collection of Industrial and Marine Bacterial

NCDO National Collection of Dairy Organism

THE END