1. Media Preparation and Sterilization

2. A medium is sterilized (living organisms removed) before usage in the lab. Sterilization methods include; autoclaving, dry-heat, filtration, UV exposure and ethylene oxide. Culture: Is part of specimen grown in culture media. Culture Media: is a medium (liquid or solid) that contains nutrients to grow bacteria in vitro. Because sometimes we cannot identify with microscopical examination directly, and sometimes we do culture for antibiotic sensitivity testing.

3. Medium is a nutrient blend used to support microbial growth
Medium is a nutrient blend used to support microbial growth. There are three physical forms of media, broth, solid, and semisolid. Solid media are more versatile in their usage. * Promote surface growth * Used to isolate pure cultures * Ideal for culture storage * Helpful in the observation of biochemical reactions * Used to make slants, deeps, and plates (named by medium)

4. Properties of Media: Support the growth of the bacteria.
Should be nutritive (contains the required amount of nutrients).
Suitable pH (neutral to slightly alkaline ).
Suitable temperature, and suitable atmosphere. (Bacteria grow at 370C)
Note: media are sterilized by autoclaving at 1210C and 1.02 atmosphere (15 p.s.i.) for minutes. With the autoclave, all bacteria, fungi, viruses, and spores are destroyed. Some media can’t be sterilized by autoclaving because they contain eggs or carbohydrates .

5. Forms of culture Solid (agar): Is Broth plus agar (seaweed).
Are prepared by adding a solidifying agent (agar %).Prepared mainly in Petri dishes, but also in tubes and slopes. After growth the bacterial colonies are visible. e.g. blood agar, chocolate agar, MacConkey agar.
Semisolid agar: (soft agar): Contains small amounts of agar ( %). Used to check for motility and also used as a transport media for fragile organisms. Can have semisolid agar in Petri dishes or in tubes. In tubes it is usually slanted to increase surface area.

6. Liquid (Broth): Mostly used for biochemical tests (blood culture, Broth culture). Growth of bacteria is shown by turbidity in medium. e.g. Nutrient broth, Selenite F broth, alkaline peptone water. Properties of agar: Some what like gelatin. It melts at 850C and solidifies at C. Comes as sold powder and then you add water to it.

7. Types of Culture Media
Simple (basal, ordinary): Culture Media: are media that contain the basic nutrients (growth factors) that support the growth of bacteria without special nutrients, and they are used as basis of enriched media. E.g. Nutrient broth, nutrient agar, peptone water. They are for the growth of non- fastidious organisms like E. coli.

8. Enriched Culture Media: are media that are enriched with: Whole blood e.g. blood agar. Lysed blood (heated to 85C) e.g. Chocolate agar. Selective Media: it is a media, which contains substances that prevent or slow the growth of microorganisms other than the bacteria for which the media is prepared for. For example EMB (Eosin Methylene blue): enteric isolation media

9. Differential Media (indicators): Contains indicators, dyes, etc, to differentiate microorganisms. E.g. MacConkey agar, which contains neutral red (pH indicator) and is used to differentiate lactose fermenter and non-lactose fermenter. (E.g. E. coli and Salmonella).

10. Common media used in Microbiology Laboratory:
Chocolate Agar: blood agar prepared by heating blood to 85C until medium becomes brown or chocolate in color heating the blood releases broth X and V growth factors and also destroys the inhibitors of V factor. These factors are required for the growth of most species of Haemophilus and also Neisseria gonorrhoear.
MacConkey Agar: an inhibitory and differential medium used to distinguish lactose- fermenting Gram- negative organism from non fermentation. Crystal violet, bile salts and neutral red are inhibitor agent. neutral red is the PH indicator.

11. Mannitol Salt Agar ( MSA ): for selective isolation for coagulase positive, mannitol-fermenting staphylococcus. Mannitol fermentation by pathogenic staphylococci is indicated by a yellow halo surrounding the colonies.Sodium chloride is the inhibitor agent.Phenol red is the PH indicator. Mueller Hinton Agar: rich medium that support the growth of most microorganism. It is commonly used for antibiotic susceptibility testing: disk diffusion antibiotic susceptibility; antibiotic serum level measurements; MBC determination.

12. Salmonella Shigella ( SS ) Agar : isolation and differential medium for pathogenic Gram-negative bacilli in particular, Salmonella and Shigella. Inhibitor for Coliforms. Triple Sugar Iron Agar (TSI): this a key medium for use in beginning the identification of a Gram- negative bacilli of the enteric group. It contains glucose (0.1% ), Lactose (1%), sucrose(1%). And peptone (2%) as nutritional sources. Sodium Thiosulfate serves as the electron receptor for reduction of sulfur and production of H2S. Detects fermentation of sucrose, lactose, glucose, as well as production of hydrogen sulfide and /or gas . Phenol red is the PH indicator; ferric ammonium citrate is H2S indicator.

13. SINGLE MEDIA / MULTIPLE TESTS

14. SINGLE MEDIA / MULTIPLE TESTS
Several media are designed to yield more than one biochemical reaction. Among the more commonly used media in this category are:
SIM medium.
Kliger's Iron agar (KIA).
Triple Sugar Iron agar (TSIA).
Lysine Iron agar (LIA).
Motility Indole Ornithine (MIO) medium.

15. SIM medium INGREDIENTS 0.4% agar ( semisolid).
Peptone ( which rich in treptophan amino acid ).
Sodium thiosulfate
Ferrous ammonium sulfate. H2S INDICATOR
The ingredients in SIM Medium enable the determination of three activities by which enteric bacteria can be differentiated.
Sodium thiosulfate and ferrous ammonium sulfate for indication of hydrogen sulfide production. The ferrous ammonium sulfate reacts with H2S gas to produce ferrous sulfide, a black precipitate.
The peptone is rich in tryptophan, which is attacked by certain microorganisms resulting in the production of indole. The indole is detected by the addition of Kovac’s reagent.
Motility detection is possible due to the semisolid nature of the medium growth radiating out from the central stab line indicates that the test
organism is motile.

16. SIM medium
Indole negative (Kovac's reagent did not turn red) and hydrogen sulfide negative (agar did not turn black).

17. SIM medium
If indole is produced from the breakdown of the amino acid tryptophan, the Kovac's reagent, when added, will turn red.

18. SIM medium
Indole negative (Kovac's reagent did not turn red) and hydrogen sulfide positive (agar turned black)

19. Kligler's Iron agar (KIA)& Triple Sugar Iron agar (TSI)
INGREDIENTS
Enzymatic Digest of Casein.
Enzymatic Digest of Animal Tissue.
Yeast Enriched Peptone.
Dextrose %.
Lactose %.
Sucrose %. NOT PRESENT IN KLIGlER’s IRON AGAR
Ferric Ammonium Citrate. AS H2S PRODUCTION INDICATOR
Sodium Chloride.
Sodium Thiosulfate.
Phenol Red. AS PH INDICATOR
Agar %.
Final pH: 7.4 ± 0.2 at 25°C.

20. PRINCIPLE
Enzymatic Digest of Casein, Enzymatic Digest of Animal Tissue, and Yeast Enriched Peptone provide the nitrogen, carbon, and vitamins required for organism growth.
Triple Sugar Iron Agar contains three carbohydrates, Dextrose, Lactose and Sucrose. When the carbohydrates are fermented, acid production is detected by the Phenol Red pH indicator.
Sodium Thiosulfate is reduced to hydrogen sulfide, and hydrogen sulfide reacts with an iron salt yielding the typical black iron sulfide.
Ferric Ammonium Citrate is the hydrogen sulfide (H2S) indicator.
Sodium Chloride maintains the osmotic balance of the medium.
Agar is the solidifying agent.

21. Results
An alkaline slant-acid butt (red/yellow) indicates fermentation of dextrose only.
An acid slant-acid butt (yellow/yellow) indicates fermentation of dextrose, lactose and/or sucrose.
An alkaline slant-alkaline butt (red/red) indicates dextrose ,lactose or/& sucrose were not fermented (non-fermenter).
Cracks, splits, or bubbles in medium indicate gas production.
A black precipitate in butt indicates hydrogen sulfide production.

22. FIG. B The small amount of acid produced in the slant of the tube during dextrose fermentation oxidizes rapidly, causing the medium to remain red or revert to an alkaline pH. In contrast, the acid reaction (yellow) is maintained in the butt of the tube because it is under lower
oxygen tension.

23. Interpretation Symbol
Results (slant/butt)
Glucose fermentation only; Peptone catabolized
K/A
Red/yellow
Glucose and lactose and/or sucrose fermentation
A/A
Yellow/yellow
No fermentation; Peptone catabolized
K/K
Red/red
No fermentation; Peptone used aerobically
K/NC
Red/no color change
Glucose and lactose and/or sucrose fermentation; Gas produced
A/A,G
Yellow/yellow with bubbles
Glucose fermentation only; Gas produced
K/A,G
Red/yellow with bubbles
Glucose fermentation only; Gas produced; H2S produced
K/A,G, H2S
Red/yellow with bubbles and black precipitate
Glucose fermentation only; H2S produced
K/A, H2S
Red/yellow with black precipitate
Glucose and lactose and/or sucrose fermentation; H2S produced
A/A, H2S
Yellow/yellow with black precipitate

25. Lysine Iron agar (LIA) INGREDIENTS Enzymatic Digest of Gelatin.
Yeast Extract.
Dextrose %.
L-Lysine %.
Ferric Ammonium Citrate. AS H2S INDICATOR
Sodium Thiosulfate.
Bromocresol Purple. AS PH INDICATOR
Agar………… %.
Final pH: 6.7 ± 0.2 at 25°C.

26. BROMOCRESOL PURPLE PH INDICATOR

27. Lysine Iron agar (LIA)

28. Motility Indole Ornithine (MIO) Medium
INGREDIENTS
Yeast Extract.
Peptone.
L-Ornithine…….. 0.5%.
Dextrose %.
Agar ……………. 0.4%.
Bromcresol Purple. PH INDICATOR
Final pH 6.5 ± 0.2 at 25°C.

29. PRINCIPLE
MIO Medium is prepared to provides three differentiating tests in one culture tube: motility, indole production, and ornithine decarboxylation. The principles of each are outlined below:
MOTILITY: Organisms are stabbed into the semisolid medium using a straight wire. If the organisms are motile, they will migrate from the stab line by means of their flagella, producing turbidity or cloudiness throughout the medium. Non-motile organisms grow only along the stab line, leaving the surrounding medium clear.
INDOLE: Tryptophan is an ingredient contained in the medium by the inclusion of peptones. If the organism possesses the enzyme tryptophanase, with the addition of Kovacs reagent, a red color will form at the top of the medium if indole is present. This reaction occurs as a result of the indole reacting with the aldehyde to yield a quinone or quinone-type structure, resulting in a red color in the alcohol layer. A negative test results in no color change.

30. PRINCIPLE continue….
ORNITHINE: The medium also tests for the presence of the enzyme ornithine decarboxylase by including L-ornithine in the agar. If the organism possesses the enzyme, it will be activated in an acid environment created by the initial fermentation of glucose. Once the amino acid is decarboxylated, the by-product diamine putrescine is produced. The result is a shift in pH to the alkaline range, turning the medium a dark purple. Organisms, which do not possess the enzyme, will stay in the acid range due to the fermentation, resulting in a yellow color in the medium.

31. BROMOCRESOL PURPLE PH INDICATOR

32. Motility Indole Ornithine (MIO) Medium