BACTERIAL IDENTIFICATION METHODS

1. BACTERIAL IDENTIFICATION METHODS CHAPTER 3

2. Content • Purification of cultures • Morphological and pure culture studies • Biochemical tests

3. 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

4. Important procedure!! Need to have a control procedure to avoid contamination. • Specimen collection • Preparation of media • Microbiological tecniques • Staining and reagents • Equipment used.

5. 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.

6. 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

7. 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 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.

8. 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

9. 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

10. Culture media Plate Broth Deep Slant

11. Morphological and pure culture studies

12. 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

13. Morphological and pure culture studies

14. Isolation of Pure Bacterial cultures Divide into 3 groups: Selective media Differental media Enrichment media

15. 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.

16. 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.

17. In MacConkey agar

18. In MacConkey agar

19. 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.

20. In nutrient agar

21. Pure colony

22. In Blood agar

23. 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) Gammahemolysis-no change in the medium.(Enterococcus faecalis)

24. Biochemical tests • Catalase test • Oxidase test • Coagulase test • Sugar fermentation test • MRVP test • Indole test • Citrate test • Motility test • H2S test • Litmus milk test

25. Catalase test • Produce bubble just after attaching the bacteria to the reagent • To differentiate staphylococci and streptococci

26. 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

27. Oxidase test

28. Coagulase test • To identify S.aureus • The enzyme coagulase clots plasma • Tube : fibrin clot • Slide: clumping of bacterial cells

29. Sugar fermentation test • Glucose test • Maltose test • Sucrose test • Lactose test • Some will appear with gas production

30. 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. (+)

31. 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

32. 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

33. Indole test using other reagent

34. 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

35. Motility test

36. 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).

38. 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.

40. Terms for today • Culture collection centre. ATCC American type culture Collection Centre NCTCCNational Collection of Type Culture NCIM Natonal Collection of Industrial and Marine Bacterial NCDO National Collection of Dairy Organism

41. The end