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

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.

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.

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.

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

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)

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.

Pure culture

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

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

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

Slant inoculation

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.

Stock culture

Liquid medium inoculation

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) .

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