1. Bacterial Cultivation and Identification
Media inoculation
@ Use sterile loops ( wire, plastic,
platinum) .
@ Also spoon, swab, pipette, capillary
tubes
@ Make intersecting lines, for discrete
colonies

3. Culture media (1). * Excess nutrients retard growth (sugars, fat,
phosphorus).
@ Types of Media:
(1) Routine media :must be:
a-Able to provide enough nutrients for
a wide range of organisms.
b-Cheap and easy to prepare.
c-Contain no C source. Bacteria convert
amino-acids in these media to energy.
* Constituents are :
a-Peptone: containing aminoacids & protein.

5. Culture Media (2) b-Inorganic salts & growth factors: add
yeast extract, blood, serum, egg yolk
C-Na Cl: added to balance osmotic pressure.
(2) Synthetic media: Materials added to give
a defined chemical composition. Used in
research studies, very expensive.
Contains: carbon source (glucose, lactose),
nitrogen source (ammonium salt), salts.
(3) Complex media: contain undefined
nutrients. Not commonly used.

7. c-Indicator media: contain chemical
Culture Media (3)
4- Special media: They include:
a- Selective media: with substances
that prevent growth of all organisms
except the one selected. Isolating a
pure strain e.g: TM medium-Neisseria.
b-Enriched media: fluid media help one
organism to multiply & prevent
growth of other organisms-Selenite-F.
c-Indicator media: contain chemical
pigments (neutral red, methylene blue)
changing colour on bacterial growth.

9. Anaerobic Culture Several techniques for anaerobic conditions:
@ Sachets containing oxygen removing
chemicals. Sachets do not produce
hydrogen, hence do not require a catalyst.
@ Copper coated steel wool to remove
oxygen. Copper absorbs oxygen, hence air
is removed from the jar.
@ Reducing agents in culture media:
* Thioglycollate broth (blood culture).
* Cooked meat medium (Clostridium culture)

11. Control of anaerobiasis: The Anaerobic jar:
@ Methylene blue as a control indicator.
The Anaerobic jar:
@ An anaerobic jar with pressure gauge,
valves, etc is not needed. A plastic air-
tight container is enough.
Carbon dioxide generation:
@ Sodium bicarbonate mixed with tartaric
acid is moistened with water.
@ Moisten Alka-Seltzer tablet with water.
@ A candle jar
@ Commercially produced sachets.

13. Reporting Cultures Sites normally sterile:
@ Report bacteria up to genera & species
Sites having a microbial flora:
@ Report bacteria for which techniques was
performed. If no pathogen isolated from
stool on a selective medium, report
No Salmonella, Shigella or Vibrio isolated
NOT No pathogen or Normal flora isolated
@ More quicker, easier, & economical to
use selective media to isolate from a
normal flora site.

15. Identification Biochemical Tests (1)
@ Analytical Profile Index (API) for all tests.
@ Bile solubility test: For S. pneumoniae
@ Catalase test: For streptococci
@ Citrate utilization test: For Entrobacteriace
@ Coagulase test: For S. aureus
@ DNA-ase test: For S. aureus
@ Indole test: Mainly for E. coli

17. Identification Biochemical Tests (2)
@ Litmus milk decolorization test: To help
identification of Enterococcus & Clostridia
@ Lysine decarboxylase test: To identify
salrnonellae and shigellae (Pos. purple).
@ Oxidase test: For Neisseria, Vibrio, Ps.
@ Urease test: For Proteus, H.pylori
@ Kligler iron agar test: For Salmonella
@ Peptone water sugars: Fermentation tests

19. Rosco Identification Tablets (1)
@ Diatabs - of Rosco Diagnostica-Denmark
@ Have a stable, long shelf-life: 2-4 years,
suitable for tropical climates, economical,
and easy to use.
@ Add tablet to a saline suspension of test
organism in a test tube, and incubate.
@ Reactions are read same day or after
four hours incubation.
@ Special reagents (supplied by Rosco) are
required to read some reactions.

21. Rosco Identification Tablets (2)
Some of the Rosco tablets tests are:
@ Beta lactamase test: To detect beta
lactamase-producing N. gonorrhoeae, etc
@ Beta galactosidase (ONPG) test: To
differentiate species of Enterobacteriacae.
@ Lysine decarboxylase test: Differentiate
between species of Enterobacteriaceae.
@ Nitrate reduction test: Detect reduction
of nitrate by Mycobacteria, Brucella, etc.
@ Ornithine decarboxylase test:
Differentiates species of Enterobacteriace

23. Rosco Identification Tablets (3)
@ Aesculin hydorolysis test: For E. faecalis
@ Factors X and V test: For H. influenzae
@ Hippurate hydrolysis test: To identify
Campylobacter, S. agalactiae
@ Voges-Proskauer (VP) test: Differentiate
between species of Enterobacteriaceae.