CHAPTER 3 BACTERIAL IDENTIFICATION METHODS

C ONTENT  Purification of cultures  Morphological and pure culture studies  Biochemical tests

P URIFICATION 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

I MPORTANT PROCEDURE !! Need to have a control procedure to avoid contamination.  Specimen collection  Preparation of media  Microbiological tecniques  Staining and reagents  Equipment used.

S PECIMEN C OLLECTION  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.

U SING 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 6

A SEPTIC T ECHNIQUE : 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 dishes inoculation of cultures These techniques include: cleaning the bench top work areas with disinfectant solution washing hands before starting work other specific techniques that will be demonstrated in the lab. 7

S TERILE 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 8

P REPARATION 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

C ULTURE MEDIA 10 Plate Slant Broth Deep

M ORPHOLOGICAL 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

M ORPHOLOGICAL AND PURE CULTURE STUDIES

I SOLATION OF P URE B ACTERIAL CULTURES Divide into 3 groups: Selective media Differental media Enrichment media

S ELECTIVE 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.

D IFFERENTIAL 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.

I N M AC C ONKEY AGAR

E NRICHMENT 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.

I N NUTRIENT AGAR

P URE COLONY

I N B LOOD AGAR

H EMOLYSIS  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)

B IOCHEMICAL TESTS  Catalase test  Oxidase test  Coagulase test  Sugar fermentation test  MRVP test  Indole test  Citrate test  Motility test  H 2 S test  Litmus milk test

C ATALASE TEST  Produce bubble just after attaching the bacteria to the reagent  To differentiate staphylococci and streptococci

O XIDASE 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

O XIDASE TEST

C OAGULASE TEST  To identify S.aureus  The enzyme coagulase clots plasma  Tube : fibrin clot  Slide: clumping of bacterial cells

S UGAR FERMENTATION TEST  Glucose test  Maltose test  Sucrose test  Lactose test  Some will appear with gas production

V OGES -P ROSKAUER 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. (+)

M ETHYL R ED 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

I NDOLE 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

I NDOLE TEST USING OTHER REAGENT

C ITRATE 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

M OTILITY 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).

T RIPLE 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 sucrose b. conduct fermentation to produce acid c. produce gas during fermentation d. generate H 2 S.

T ERMS 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

T HE END