By Dr. Shnyar Hamid Qadir Culture Media Lab.3 By Dr. Shnyar Hamid Qadir

A microbiological medium (media, plural) is the food that we use for culturing bacteria, molds, and other microorganisms. It can exist in three consistencies: Liquid Solid Semisolid.

Liquid media Include nutrient broth, citrate broth, glucose broth, litmus milk,…. These media are used for the propagation of large numbers of or­ganisms, fermentation studies, and various other tests.

Solid media Are made by adding a solidifying agent, such as agar, gelatin, or silica gel, to a liquid medium. A good solidifying agent is one that is not utilized by microorganisms, does not inhibit bacterial growth, and does not liquefy at room temperature

Nutrient agar, and blood agar, are examples of solid media that are used for devel­oping surface colony growth of bacteria. As we will see, the develop­ment of colonies on the surface of a medium is es­sential when trying to isolate organisms from mixed cultures.

Semisolid media Fall in between liquid and solid media. Although they are similar to solid media in that they contain solidifying agents such as agar and gelatin, they are more jellylike due to lower percent­ages of these solidifiers. These media are particularly useful in determining whether certain bacteria are motile

NUTRITIONAL NEEDS OF BACTERIA Any medium that is to be suitable for a specific group of organisms must take into account the following seven factors: water carbon energy nitrogen minerals growth factors and pH

Isolation and Identification of Bacteria Culture A useful feature of agar plates is that the bacteria can be separated by spreading a small sample of the specimen over the surface. Bacterial cells that are well separated from others grow as isolated colonies, often reaching 2 to 3 mm in diameter after overnight incubation. This allows isolation of bacteria in pure culture because the colony is assumed to arise from a single organism Colonies vary greatly in size, shape, texture, color, and other features called colonial morphology.

Bacteriologic plate streaking

Bacteriologic plate streaking Plate streaking is essentially a dilution procedure. A. (1)The specimen is placed on the plate with a swab, loop, or pipette and evenly spread over approximately part of plate surface with a sterilized bacteriologic loop.(2-5) The loop is flamed to remove residual bacteria, and a series of overlapping the streaks are made flaming the loop between each one. B. After overnight incubation, heavy growth is seen in the primary areas followed by isolated colonies.

Culture Media Over the last 100 years, countless media have been developed by microbiologists to aid in the isolation and identification of medically important bacteria and fungi. Only a few have found their way into routine use in clinical laboratories. These may be classified as Nutrient Selective Indicator media.

Nutrient Media The nutrient component of a medium is designed to satisfy the growth requirements of the organism to permit isolation and propagation. These media are prepared with enzymatic or acid digests of animal or plant products such as muscle, milk, or beans.

Nutrient Media The digest reduces the native protein to a mixture of polypeptides and amino acids that also includes trace metals, coenzymes, and various undefined growth factors. For example, one common broth contains a pancreatic digest of casein (milk curd) and a papaic digest of soybean meal. To this nutrient base, salts, vitamins, or body fluids such as serum may be added to provide pathogens with the conditions needed for optimum growth.

Selective Media Selective media are used when specific pathogenic organisms are sought in sites with an extensive normal flora (eg, N gonorrhoeae in specimens from the uterine cervix or rectum). In these cases, other bacteria may overgrow the suspected etiologic species in simple nutrient media, either because the pathogen grows more slowly or because it is present in much smaller numbers. Selective media usually contain dyes, other chemical additives, or antimicrobics at concentrations designed to inhibit contaminating flora but not the suspected pathogen.

Indicator Media Indicator media contain substances designed to demonstrate biochemical or other features characteristic of specific pathogens or organism groups. The addition to the medium of one or more carbohydrates and a pH indicator is frequently used. A color change in a colony indicates the presence of acid products and thus of fermentation or oxidation of the carbohydrate by the organism. The addition of red blood cells (RBCs) to plates allows the hemolysis produced by some organisms to be used as a differential feature.

In practice, nutrient, selective, and indicator properties are often combined to various degrees in the same medium. It is possible to include an indicator system in a highly nutrient medium and also make it selective by adding appropriate antimicrobics.

Atmospheric Conditions -Aerobic Once inoculated, cultures of most aerobic bacteria are placed in an incubator with temperature maintained at 35° to 37°C. Slightly higher or lower temperatures are used occasionally to selectively favor a certain organism or organism group.

Aerobic Most bacteria that are not obligate anaerobes grow in air; however, CO2 is required by some and enhances the growth of others. Incubators that maintain a 2% to 5% concentration of CO2 in air are frequently used for primary isolation, because this level is not harmful to any bacteria and improves isolation of some. A simpler method is the candle jar, in which a lighted candle is allowed to burn to extinction in a sealed jar containing plates. This method adds 1% to 2% CO2 to the atmosphere.

Anaerobic Strictly anaerobic bacteria do not grow under the conditions just described, and many die when exposed to atmospheric oxygen. Most medically important anaerobes grow in the depths of liquid or semisolid media containing any of a variety of reducing agents, such as cysteine, thioglycollate, ascorbic acid, or even iron filings. An anaerobic environment for incubation of plates can be achieved by replacing air with a gas mixture containing hydrogen, CO2, and nitrogen and allowing the hydrogen to react with residual oxygen on a catalyst to form water.

Commonly Used Bacteriologic Media

Nutrient broths. Some form of nutrient broth is used for culture of all direct tissue or fluid samples from sites that are normally sterile to obtain the maximum culture sensitivity. Selective or indicator agents are omitted to prevent inhibition of more fastidious organisms.

Blood agar. The addition of defibrinated blood to a nutrient agar base enhances the growth of some bacteria, such as streptococci. This often yields distinctive colonies and provides an indicator system for hemolysis.

Two major types of hemolysis are seen: β-hemolysis, a complete clearing of red cells from a zone surrounding the colony And α-hemolysis, which is incomplete (ie, intact red cells are still present in the hemolytic zone), but shows a green color caused by hemoglobin breakdown products. The net effect is a hazy green zone extending 1 to 2 mm beyond the colony. A third type, α'-hemolysis, produces a hazy, incomplete hemolytic zone similar to that caused by α-hemolysis, but without the green coloration.

Blood agar

Chocolate agar. If blood is added to molten nutrient agar at about 80°C and maintained at this temperature, the red cells are gently lysed, hemoglobin products are released, and the medium turns a chocolate brown color. The nutrients released permit the growth of some fastidious organisms such as Haemophilus influenzae, which fail to grow on blood or nutrient agars. This quality is particularly pronounced when the medium is further enriched with vitamin supplements.

Chocolate agar

Martin–Lewis medium. A variant of chocolate agar, Martin–Lewis medium is a solid medium selective for the pathogenic Neisseria (N gonorrhoeae and N meningitidis). Growth of most other bacteria and fungi in the genital or respiratory flora is inhibited by the addition of antimicrobics includes vancomycin.

MacConkey agar. This agar is both a selective and an indicator medium for Gram-negative rods, particularly members of the family Enterobacteriaceae and the genus Pseudomonas. In addition to a peptone base, the medium contains bile salts, crystal violet, lactose, and neutral red as a pH indicator. The bile salts and crystal violet inhibit Gram-positive bacteria and the more fastidious Gram-negative organisms, such as Neisseria. Gram-negative rods that grow and ferment lactose produce a red (acid) colony, often with a distinctive colonial morphology.

MacConkey agar

Hektoen enteric agar. The Hektoen medium is one of many highly selective media developed for the isolation of Salmonella and Shigella species from stool specimens. It has both selective and indicator properties. The medium contains a mixture of bile, thiosulfate, and citrate salts that inhibits not only Gram-positive bacteria, but members of Enterobacteriaceae other than Salmonella and Shigella that appear among the normal flora of the colon.

Hektoen enteric agar

Anaerobic media. In addition to meeting atmospheric requirements, isolation of some strictly anaerobic bacteria on blood agar is enhanced by reducing agents such as L-cysteine and by vitamin enrichment. Sodium thioglycolate, another reducing agent, is often used in broth media. Plate media are made selective for anaerobes by the addition of aminoglycoside antibiotics, which are active against many aerobic and facultative organisms but not against anaerobic bacteria. The use of selective media is particularly important with anaerobes because they grow slowly and are commonly mixed with facultative bacteria in infections.