PHT 226 Lab# 4

1. PHT 226 Lab# 4 • Culture media • Streaking

2. Culture Media • The culture media is an artificial preparation contains the essential element and nutrient in a proper concentration needed by the microorganism to grow. • It may be:- • Liquid (broth) • Solid (containing agar) • Semisolid (containing low conc of agar)

3. Culture Media • Most common ingredients:- • Essential elements and nutrients. • Solidifying agents.

4. Objectives of bacterial culture: • To study bacterial morphology, physiology and pathogenic properties. • At the clinical level: • Isolation of bacteria in pure culture • Identification • Antimicrobial susceptibility • Preparation of important bacterial products, e.g., toxins, antigens, vaccines … etc.

5. Essential Elements and Nutrients • A Source of Nitrogen: • (peptone) • meat extract, • yeast extract, • amino acids & in organic compound.

6. Essential Elements and Nutrients • A Source of Carbon: • usually supplied in form of carbohydrates. • Inorganic salts: • normally present in peptone but added in traces [e.g; Na, K, Ca, Mn, Fe, Co, Zn…….etc] • NaCl

7. Essential Elements and Nutrients 4.Buffers: • added to resist any changed in pH of the medium. • Blood and milk are naturally occurring buffers.

8. Solidifying Agents • Agar: it is the most common solidifying agent, used in conc. * 1.5 – 2% ( solid medium). * 0.2 – 0.5% (semisolid). ✍ it extracted from certain red marine algae, and composed of complex carbohydrates not easily broken down by most common bacteria.

10. Solidifying Agents 2.Gelatin: • it is protein derivative added to nutrient broth in conc. of 12 -15% as a test for proteolytic activity of bacteria.

11. Classification of Media • According to its content:- • Non synthetic complex medium; • contains extracts or digests of plant or animal tissues such as beef, peptone & soybean digest.

12. Classification of Media • Synthetic Chemically defined medium; • contains known chemical compounds such as: • amino acids, • sugar, • Vitamins & salts.

13. Classification of Media • Living medium; • it is a living cell of animal or plant in which m.o could be cultivated such as viruses (intracellular tissue culture.)

14. Types of culture media: • Simple media • Enriched media • Selective media • Indicator media • Selective and indicator media

15. Classification of Media • According to the purpose of use:- 1- Simple [Basic] media; • contain only the essential or basic nutrients which support the growth of most common bacteria. ✎ e.g; • Nutrient agar (plate, slant) • Nutrient broth

18. Classification of Media 2- Enriched media; • in addition to basic nutrients, it contains enriched substances such as blood, serum, vitamins to support the growth of most pathogens can’t grow on ordinary simple media. ✎e.g; • blood agar, • Chocolate agar, • Serum agar.

19. Blood agar

20. Chocolate agar(Neisseria spp.) C.diphtheria

21. Classification of Media 3.Selective media; • mostly used in case of mixture. • Contains substances which selectively enhance the growth of certain particular species and inhibit the growth of undesired bacterial species. • These inhibitory substances include: dyes, heavy metals, chemicals, antimicrobial agents …

22. Examples of Selective Media • Mannitol salt agar; • it is selective medium for Staphylococcus species because it contains high conc. of salt (about 7%).

23. 2- Lowenstein Jensen medium it is selective medium for M. tuberculosis; • because it contains malachite green dye. • It contains also eggs which solidify the medium without agar and make it enriched medium.

25. Examples of Selective Media 3- MacConkey agar: • it is selective medium for gram –vebacteria because it contains bile salt which inhibit the growth of gram +ve bacteria.

26. Examples of Selective Media 4- Cetrimide agar: • it is highly selective medium for pseudomonas species. It contains Cetrimide. • Cetrimide is a surfactant that inhibit the growth of all organism except pseudomonas.

27. Classification of Media 4- Indicator media: • Contain substances which are affected by the growth of organism to produce characteristic reaction such as indicators which indicates acid production during sugar fermentation.

28. Examples of Diagnostic Media • Mannitol salt agar; • It contains mannitol and phenol red indicator. • S.aureus is the only strain of staphylococcus species can ferment mannitol, leading to production of acid which can detected by yellow color around the growth resulting from changing the color of the indicator.

29. Growth with yellow color around the colonies S.aureus • Growth without change in the color of the medium S.epidermidis

30. Examples of Diagnostic Media 2.MacConkey agar; • Gram –ve bacteria classified to: • It contains neutral red indicator which detect acid production due to fermentation of lactose which done by lactose fermenter gram –ve bacteria such as E-coli and Klebsiella species to give rose pink colonies. Lactose fermenter Lactose non-fermenter

31. Lactose fermenter Pink colonies • Lactose non-fermenter Pale colonies

32. Classification of Media 5.Selective and indicator media; • are both selective and diagnostic since they permit the growth of a few desired species only & contain indicator which enable us to differentiate between the species which are able to grow on these media. • e.g.; Mannitol salt agar, MacConkey agar.

33. Media for Fungi ❀Sabouraud dextrose agar. similar in composition as those of bacteria EXCEPT: • They are acidic in nature (PH 3 -6). • contain readily fermentable sugars e.g.; dextrose and maltose. ☞ Culture of Fungi usually incubate at lower temperature than bacteria (about 25°C).

34. Anaerobic media • Fluid thioglycolate: It is anaerobic medium due to presence of: • Sodium thioglycolate and cystaine which act as reducing agents. • Cooked meat medium: It is anaerobic medium due to presence of: • Meat particles (prepared from heart muscles) • Hematin& glutathione in meat particles act as reducing agents.

35. Anaerobic media

36. Anaerobic Jar - The nutrient agar plates employed for routine culturing do not contain reducing agents. - They can be used to culture anaerobes if they are placed inside an anaerobe jar ( a chamber from which the oxygen is removed).  - Gas generator packets (e.g. GasPak) are placed in the jar along with plates or tubes containing conventional media destined to be incubated anaerobically.  - In order to assure that oxygen actually was removed from the chamber, a strip of paper soaked in methylene blue dye is included in the jar.  - It is blue when exposed to oxygen but will become colorless (white) when oxygen is absent. 

37. GAS PAK SYSTEM FOR ANAEROBIC CULTIVATION

38. Colony vs. Cell Cells Colonies

39. Introduction to Microbiological Equipments and Materials • Inoculation: Culturing of sterile media with m.o [Inoculation loop]. • Incubation: Placing the culture into the incubator at optimum temperature for growth. • Sterile: Free from any living microorganism • Contamination: Introduction of undesirable m.o.

40. Description of Colonies

41. Isolation of Pure Colonies of Microorganism “Streak Plate Method” In natural environments, bacteria & other m.o exist in mixed population. “Streak Plate Technique” • The individual cells are separated from each other by certain distance on the surface of the agar. • After incubation, each single deposited cell divide many times and finally form visible mass of growth “COLONY”.

42. The streak Plate Method • The culture prepared from a single type of colony is regarded as a pure culture. • The streak Plate Method is used for: • Checking the purity of a bacterial culture. • Isolating individual species from a mixture culture.

44. The streak Plate Method • Objective:- for isolation of individual species of a mixed broth culture. • Materials:- • Nutrient agar plate. • Mixed broth culture of Serratia marcescens and staph. aureus.

45. S & S The streak Plate Method • Procedure: Drop of the culture Flame & Cool Flame & Cool Flame & Cool Aseptic technique Invert the plate and Incubate for 24h at 37℃

47. The streak Plate Method

48. The streak Plate Method

49. Sources of Contamination

50. Sources of Contamination • Objective: To identify some of the sources of contamination present in the lab. ✔in order to avoid them • Contamination from hands. • Contamination from breath. • Contamination from air. • Contamination from bench.