1. Handling of Bacterial Cultures
Practical No. 3
Handling of Bacterial Cultures
Department of Microbiology
College of Medicine

3. Microscopic examination of microorganisms provides information about their morphology but does not tell us much about their biological characteristics. To obtain information about the latter it is necessary to observe microorganisms in culture.
If we are to cultivate them successfully in the laboratory, we must provide them with suitable nutrients, such as protein components, carbohydrates, minerals, vitamins, and moisture in the right composition. This mixture is called a culture medium (plural, media). It may be prepared in liquid from, as a broth, or solidified with agar (a nonnutritive solidifying agent extracted from seaweed). Agar media may be used in tubes as a solid column or as slants. They are also commonly used in Petri dishes (named for the German bacteriologist who designed them) or plates, as they are often called.

4. Solid media are essential for the isolation and separation of more than one type of bacteria growing together in a specimen( called mixed culture). If a mixture of bacteria is spread across the surface of a plated agar medium, individual organisms will multiply at individual sites until a visible aggregate called a colony is formed. One colony of a single species can then be separated from the rest and transferred to another medium, where it will grow as a pure culture, and can be studied as such.
In liquid media, some bacteria may grow diffusely, producing uniform clouding. Others may look very granular in broth, layering of growth at the top, center, or bottom of a broth tube reveals something of the organism's oxygen requirements.. Observation of such features, and others, can also be helpful in recognizing types of organisms.

5. Bacterial growth patterns
In liquid medium:
Superficial growth;
Turbidity/diffuse;
Precipitate growing;
(sediment)
In solid medium:
Confluent growth / Smear;
Colony:
a cluster of microorganisms growing
on a solid medium. It is directly visible
and arises from a single cell.

6. In semi-solid medium:
Only grow along the line of inoculation
Grow diffusely

7. Student must learn how to handle cultures aseptically
Student must learn how to handle cultures aseptically. The organisms must not be permitted to contaminate the worker or the environment and the cultures must not be contaminated with extraneous organisms.
It would be advisable, before you begin, to reread the opening paragraphs of part one dealing with safety procedures and general laboratory directions.
Objective
To make aseptic transfers of pure cultures and to examine them for important gross features.
Materials;
- tubes with nutrient broth
- slants of nutrient agar
- different cultures of organisms
- Wire inoculating loop
- Bunsen burner (and matches)
- China-marking pencil (or labels)

8. Definition of pure culture:
It is a single kind of microorganisms growing alone in a protected environment. Media and equipment to be used must be sterile. And technique must be aseptic to avoid contamination from sources other than the one being investigated.

9. Pure culture

10. General procedure of bacteriological Diagnosis
Culture medium
concept
categories
bacterial growth patterns
Inoculation and transfer techniques

11. Inoculation and Transfer Techniques
Streak-plate technique
Slant inoculation
Liquid medium inoculation
Semisolid medium inoculation

13. Streak-plate technique
four-area streak plate technique I
1/10 II I
1/5
Rotate plate 90
Flame loop
Flame loop
Rotate 90
III
1/4 IV
Rotate 90

16. Slant inoculation

17. Stock culture:
Stock culture means the preservation of a microorganism in a culture medium for future study. It can be kept usually for about one month at 4°C.
Technique of stock culture:
Use a universal bottle containing medium in a slanted position ( to provides a wide surface for inoculation and good nutrition).
Inoculate the surface of the medium with few colonies of a pure culture.
Incubate at 37°C for 24 hours, then store at 4°C.

18. Stock culture

19. Liquid medium inoculation

20. Cultural characteristics (Colonial morphology)
Macroscopic characteristics of a pure culture: Size: measured in m.m. Shape: punctiform, circular, filamentous, irregular, spindle. Elevation: flat, raised, convex, umbonate. Margin: entire, lobate, filamentous. Consistency: dry, mucoid. Surface texture: smooth, rough Color or pigmentation: yellow, green … Opacity: Opaque, translucent, glistening. Odour : bad odour, sweat musty odour. Changes in the inoculated medium (haemolysis, bile precipitation) .

21. Thank You