culture-media-and-methodsppt-256669261.pptx

Microbiology

1. Culture Media C unsure Methods Culture Media used in IYlicrobiology

2. The food material or substances required for growing microorganisms in vitro (outside the body) is called as Culture medium. Culture Media used in Microbiology

3. Important to grow mas. outside the body for following purposes:- - To Identify the cauae of infection from clinical samples H so proper treatment can be given. — To study characteristics or properties of m.os. To prepare biological products like Vaccines ToxoldeB Antigens.......etc.

4. Water Energy source Carbon source Nitrogen source Mineral salts Special growth factors

5. Semi- Solid Media Liquid Media l. Based on their consistency

6. Distinct colony morphology Characteristics — cosy to rTentif Colony — macroscopically visible collection of millions of bacteria originating from a single bacterial cell

7. Diffused growth No characteristics for identification Difficult to isolate Earliest liquid medium: urine or meat brotn used by Louis Pasteur. Also referred to as "Broth”. E

8. Earliest solid medium: Cooked cut potato by Robert Koch Gelatin - not satisfactory - liquefy at 24•C Frau Heaee Universally used for preparing solid medium Obtained from seaweed: Gelidium No nutritive value Not affected by the growth of the bacteria. Melts at 85•C & solidifee at 4Z°C. 2% agar lg employed in solid medium

9. l. Based on their consistency Solid medium Liquid medium Semi solid medium II. Based on the constituents/ ingredients Simple medium Complex medium Synthetic or defined medium Special media

10. Moet a›rnrnon in routine diagnostic laboratories Eg: Nutrient Broth, Nutrient A 8 ® * NB consists of peptone, meat extract, NaCI, water “ ’” +’"’’* “’”" ”’’’"s” NB * 2% agar = Nutrient agar Agar conc. Reduced (0.2 0.5%) = Semi- solid medium

11. Media other than basal media. They have added complex ingredients such as yeast extract or casein hydrolysa” which consist of a mixture of many chemical species in unknown proportions. Provide special nutrients Synthetic or defined ‹riedia Media prepared from pure chemical substances exact composition is known Used for special studies, eg. metabolic requirements Eg: peptone water- (1% peptone + 0.5% NaCI in water)

12. Substances like blood, serum, egg are added to the basal medium. Used to grow Fastidious organisms. Eg: Blood agar, Chocolate agar

13. m Preparation Blood Agar - 3• N.A. + 5% Sheep Blood N.A. autoclaved 6 cooled to 45*C 4 add 5% sheep blood 6 allow to solidity. mix 6 E.g for the growth of fastidious organisms and detect hemolysis e.g. Streptocaccus pyogens streptococcus pneumoniae Nisseria meningitidis Choclate agar U N.A. + 5% sheep blood — N.A. autoclaved 1 allow it to solidi same as above cooled to 72*C 1 add 5 A sheep blood mix 1 Heamophilus influenzae

14. Liquid media used to isolate pathogens from a mixed culture. Stimulate growth of desired bacterium Inhibit growth of unwanted bacterium Media is incorporated with inhibitory substances to suppress the unwanted organism — • increase in numbers of desired bacteria — Selenlte F Broth — for the isolation of Salmonella, Shlgalta - T trathlonate Broth - inhibit ooliforms — Alkaline Peptone Water - for li8rio cfio/e ae

15. Tetrath i oate Al ka It \e Pepto\ e wa*er

16. The inhibitory substance is added to a solid media to Inhibit commensal or contaminating bacteria e.g. Antibiotics Chemicals Alteration pH Increaee in number of colonies of desired bacterium e.g: Deoxycholate nitrate medium for dysentery bacilli Igac Conkey'a madium for gram negative bacteria. T B I m - gf. tubercu/oefs

17. Nyatattn — Trlmethoprim Selective media for Neisseria gonorrhoeae Usuaily contains combination - Vacomycin Coliatin Rectsl Specimen

18. Thiosulfate citrate

19. LJ m ed i a

20. Contain an indicator which Ba uoIoza'when a bacterium grows in them e.g.: WlIaon•BIsir uzedlum - S. typh/ flames black cafoofes Mclmod's medium (Potassium te1lurite}- Diphtharla bacilll

21. shows three types of Hemolysis a Hemolysis t Hemolysis y Hemolysis Æpha Hœnołysls

22. Substances incorporated in it enabling it to distinguish between bacteria. Eg: Mac Conkey's medium Peptone Lactose Agar Neutral red Taurocholate Distinguish between lactose fermenters (Pink colour) & non lactose fermenters (C olourless).

23. MacConkey agar Lactose fermenters — Pink colonies Non lactose fermenters — Colourless colonies

24. Media containing any fermentable substance Eg: glucose, arabinose, lactose, starch etc. Media consists: 1% of the sugar in peptone water + Indicator Contain a small tube (Durham's tube) for ttie detection of gas by the bacteria

25. Media used for transporting the samples. Delicate organisms may not survive the time taken for transporting the specimen without a transport media Eg: Stuart's medium — non nutrient soft agar gel containing a reducing agent & charcoa! used for Goi›nOcOccl Buffered glycerol saline — enteric bacilli

26. These media are used to grow anaerobic organisms. Eg: Thioglycolate medium — Robertson's cooked meat medium Anaerobic Culture Media ,

27. Culture methods employed depend on the purpose for which they are intended. — To isolate bacteria in pure cultures. To demonstrate their properties. To obtain sufficient growth for the preparation of antigens and for other tests. To determine sensitivity to antibiotics. To estimate viable counts. - Maintain stock cultures.

28. Lawn culture Stroke culture Stab culture Pour plate method Liquid culture Anaerobic culture methods

29. Used for the isolation of bacteria in pure culture from clinical specimens. Platinum wire or Nichrome wire is used. One loopful of the specimen is transferred onto the surface of a well dried plate. Spread over a small area at the periphery. The inoculum is then distributed thinly over the plate by streaking it with a loop in a series of parallel lines in different segments of the plate. On incubation, separated colonies are obtained over the last series of streaks.

30. Primary - inoculum Fig. 2.5.1 Streak culture (surface plating) on solid medium

31. Provides a uniform surface growth of the bacterium. Us66 - Antibiotic sensitivity testing. In the preparation of bacterial antigens and vaccines. own cultures are prepared by flooding the surface of the plate with a liquid suspension of the bacterium.

32. Antibiotic Sensitivity Testing

33. Stroke culture is made in tubes containing agar slope / slant. Uses Provide a pure growth of bacterium for slide agglutination and other diagnostic tests.

34. Prepared by puncturing a suitable medium — gelatin or glucose agar with a long, straight, charged wire. Uses Demonstration of gelatin liquefaction. Oxygen requirements of the bacterium under study. Maintenance of stock cultures.

35. Agar medium is melted (15 ml) and cooled to 45 C. 1 ml of the inoculum is added to the molten agar. Mix well and pour to a sterile petri-dish. Allow it to set. Incubate at 37 o C, colonies will be distributed throughout the depth of the medium. Uses Gives an estimate of the viable bacterial count in a suspension. For the quantitative urine cultures.

36. Liquid cultures are inoculated by touching with a charged loop or by adding the inoculum with pipettes or syringes. Uses Blood culture Sterility tests Continuous culture methods Disadvantage — It does not provide a pure culture from mixed inocula.

37. ANAEROBIC CULTURE METHODS Anaerobic bacteria differ in their requirement and sensitivity to oxygen. CI. tetani in a strict anaerObe. Methods: Production of vacuum Displacement of oxygen with other gases Chemical method Biological method Reduction of medium

38. Production of vacuum: Incubate the cultures in a vacuum desiccators. Displacement of oxygen with other gases Displacement of oxygen with hydrogen, nitrogen, helium or CO . Eg: Candle jar

39. Chemical method Alkaline pyrogallol absorbs oxygen. Chromium and Sulphuric acid.

40. 3¢nfz›B›a8 - PPdas'aoae eéfe Consists of a metal jar or glass jar with a metal lid which can be clamped air tight. The lid has 2 tubes — gas inlet and gas outlet Tha lid has two terminals — connected to electrical supply Catalyst :- Aluminium Pallets coated with pallidium

41. Mclntosh and Fildes’ Anaerobic jar

42. Act as a c at aT y s I for th c c a \æb \ ri at a I of hyd ogen with res id t\a I oxyge n Working

43. Commercially available disposable packets containing Ilets of Sodium borohydride, cobalt chloride, citric acid and sodium bicarbonate. Contains chemicals which generate 2 and CO 2 on addition of water. Cold catalyst — permits combination of Hydrogen & Oxygen Indicator is used reduced methylene blue. Colourless — anaerobically — Blue colour— on exposure to oxygen

44. Reduction Agents Oxygen in culture media U reduced by various agents e.g. thioglycollate, cooked meat pieces, cysteine and ascorbic acid. By using reducing agents 1% Thioglycolate. Robertson's cooked meat (RCM) medium.