Chapter 2: Culture microbiological specimen

Culture of microbiological specimens is a fundamental technique in applied biology that enables the growth, identification, and analysis of microorganisms from various samples. This process is essential for diagnosing infections, monitoring environmental samples, and supporting research in clinical, agricultural, and industrial settings. In Kenya, professionals in hospitals, research laboratories, and food safety agencies rely heavily on culture methods to detect pathogenic organisms, ensure public health safety, and support disease control initiatives. Mastery of culture media and reagents is critical for successful cultivation and accurate interpretation of microbial growth.

2.1 Culture Media and Reagents

A culture medium is a prepared mixture of nutrients in which microorganisms can grow outside their natural habitat. Reagents are the other chemicals and solutions used with it to dilute, buffer, indicate, supplement or select. The composition and the physical state of the medium decide which organisms grow, how fast they grow and how they look, so the medium is chosen to match the organism and the purpose of the test. Example: a water testing laboratory that wants to count bacteria in a borehole water sample uses a diluent, a solid plate medium and sterile pipettes, and each must be correct for the count to be valid.

Components of culture media

• Water: distilled or deionised water, because tap water carries minerals, chlorine and microbes that change the medium.
• Carbon and energy source: sugars such as glucose, lactose or other carbohydrates.
• Nitrogen and amino acids: peptone and tryptone, made from digested protein, and meat or yeast extract, which supplies vitamins and growth factors.
• Salts and buffers: sodium chloride for osmotic balance and phosphates to keep the pH steady.
• Solidifying agent: agar, a polysaccharide from red seaweed that most bacteria cannot digest, so it stays solid during growth.
• Indicators: pH indicators such as phenol red and neutral red that change colour when bacteria produce acid or alkali.
• Supplements and selective agents: blood, serum or egg yolk for demanding organisms, and bile salts, dyes or antibiotics to hold back unwanted bacteria.

Defined and complex media

A defined (synthetic) medium has a known chemical composition, with every ingredient measured exactly. It is used in research on nutritional requirements and metabolism. A complex medium contains digests or extracts such as peptone and yeast extract, whose exact composition varies, and it supports a wider range of organisms and is used for routine work.

Reagents used with culture media

• Diluents: sterile saline or peptone water for serial dilution of samples.
• Buffered diluents: phosphate-buffered saline or buffered peptone water keep the pH steady when a sample is diluted.
• Sterile water: water that has been autoclaved, used to make up diluents and to rinse glassware before it is sterilised.
• Heat-sensitive additives: serum, some antibiotics and similar supplements are sterilised separately and added after the base medium has cooled, as the label directs.
• Staining and test reagents: reagents such as Gram's iodine, Kovac's reagent and hydrogen peroxide are used for staining and identification and are described under Bacterial Identification.

How media are weighed, sterilised, stored and checked is covered under Media Preparation.

2.1.1 Forms of Culture Media

The physical state of the medium decides how organisms grow and what can be seen. The three forms differ in the amount of agar they contain, and each answers a different question.

2.1.1.1 Broth

Broth is a liquid medium with no agar. Microorganisms multiply throughout the liquid and growth is seen as cloudiness, a ring or film at the surface, or a deposit at the bottom of the tube. It gives a large number of cells quickly and is easy to sample.

• Uses: enriching a sample that contains few of the target organisms, preparing an inoculum for a test, and carrying out biochemical tests in tubes.
• Examples: nutrient broth, peptone water and tryptone broth.
• Aeration: shaking the culture supplies oxygen to aerobic organisms and gives faster growth.
• Limit: broth does not separate organisms, so a mixed culture in broth cannot be separated until it is streaked onto a solid medium.

2.1.1.2 Solid

A solid medium contains agar, which melts at about 85 to 95 °C and sets as the medium cools to about 40 °C. Organisms placed on the surface grow into visible colonies, and each colony usually comes from a single cell or a small group of cells.

• Uses: isolating pure cultures, counting colony-forming units in food and water samples, and studying the colour, shape, size and edge of colonies.
• Examples: nutrient agar, MacConkey agar and blood agar.
• Formats: plates in Petri dishes, slopes in tubes for storing cultures and deep tubes for oxygen studies.
• Handling: plates are dried before use, stored upside down and incubated upside down to prevent condensation running over the colonies.

2.1.1.3 Semi-solid

A semi-solid medium contains only a small amount of agar, so it is soft and gel-like. It is usually dispensed into tubes and inoculated by stabbing straight down the centre.

• Motility testing: motile bacteria swim away from the stab line and make the medium cloudy, whereas non-motile bacteria grow only along the line. Proteus is motile and Klebsiella is not, so the test helps to tell them apart.
• Oxygen requirement: growth only near the top suggests aerobic organisms, growth throughout the tube suggests organisms that grow with or without oxygen, and growth only deep in the tube suggests anaerobic organisms.
• Transport: soft agar media are used to keep specimens alive during transport, as described under Transport Media.
• Interpretation: results must be read against a control tube, and the stab must be straight, because a crooked stab can be mistaken for spread.

Chapter Summary

This chapter explored the fundamental aspects of culturing microbiological specimens, beginning with an overview of culture media and reagents, including the three primary forms: broth, solid, and semi-solid media. It then detailed the various types of culture media such as basic, transport, selective, non-selective, enriched, and enrichment media, each serving distinct roles in microbial growth and isolation. The process of media preparation was discussed to emphasize the importance of proper formulation and sterilization techniques. Several inoculation methods were examined, including pouring, streaking, stabbing, and spreading, which are essential for introducing microorganisms onto or into culture media. The chapter also covered the use of slopes and deep cultures as specialized techniques for maintaining and growing bacterial cultures. Finally, bacterial identification methods were outlined, focusing on staining techniques like Gram stain and acid-fast staining, as well as biochemical tests that help characterize and differentiate bacterial species.