Culturing bacterial Pathogens

Pathogenic Bacteria Pathogenic bacteria are bacteria that cause bacterial infections in human beings, animals and plants. Pathogenic bacteria contribute to globally important diseases, such as pneumonia, which can be caused by bacteria such as Streptococcus and Pseudomonas and Food borne illnesses, which can be caused by bacteria such as Shigella, Campylobacter and Salmonella.

Inoculated media should be incubated as soon as possible. WHY? Delay in incubation can affect the viability of pathogens. Increase the risk of plates becoming contaminated. Microorganisms require incubation at the temperature, in the humidity and gaseous atmosphere most suited to their metabolism.

Temperature of incubation

Most of pathogenic bacteria are Mesophiles i.e. Middle/Moderate temperature loving (Neither too hot nor too cold). Growth is in between 25 to 40 o C. Optimum temperature is commonly 37 o C. Many have adapted to live in the bodies of animals.

In laboratory we incubate inoculated culture media in a closed instrument with a temperature control, known as an incubator.

Humidity during incubation While incubating agar plates, make sure to keep an open beaker of water in the incubator. Periodically check that the beaker has water in it - do not let it run dry. The water will maintain a constant level of humidity in the incubator. Most of modern incubators have build in water reservoirs by which humidity in the chamber can be regulated. Gonococci are rapidly killed in dry conditions

Why humidity in incubator……….? Humid environment inside incubator will help in uniform transfer of heat through out the incubator. Moist condition inside an incubator will also tend to minimize drying out of agar media in plates, test tubes or screw cap bottles. Culture media tend to deteriorate more rapidly when undue drying occurs. Most organisms grow maximally when humidity is 70% or higher e.g. H. pylori and N. gonorrhoeae require an atmosphere with high humidity for their growth.

Presence of O 2 and CO 2 in incubator Requires free oxygen to survive. Pseudomonas aeruginosa. Obligatory aerobe Grows best in the presence of only a trace of free oxygen. Campylobacter jejuni. Microaerophilic Survives only in the absence of oxygen. Clostridium tetani. Obligatory anaerobe Can live with or without free oxygen. Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli. Facultative anaerobe Requires an atmosphere which contains carbon dioxide. Neisseria meningitidis. A carboxyphilic organism Can’t use oxygen, but tolerate its presence e.g. Lactobacillus Aerotolerant Anaerobes Grow better at high CO2 levels and low O2 levels. Similar to environment of intestinal tract e.g. Clostridium Capnophiles According to the gaseous requirements microorganisms are:

The Requirements for Growth: Related to Oxygen Oxygen (O 2 ) Dr.T.V.Rao MD 11 Table 6.1

Culturing of anaerobes Aim 1. An anaerobic atmosphere is essential for the growth of strict anaerobes such as Clostridium species. 2. Anaerobic incubation also helps to differentiate pathogens and to isolate facultative anaerobes from specimens containing commensals, e.g. Streptococcus pyogenes from throat swabs. 3. The haemolytic reactions of beta-haemolytic streptococci are also more pronounced following anaerobic incubation.

Methods for obtaining anaerobic conditions 1.Commercially produced kits containing oxygen removing chemicals. 2.Reducing agents in culture media.

I. Commercially produced kits The GasPak Anaerobic System is used to create an oxygen-free environment for the growth of anaerobic microorganisms. Inoculated plates or tubes are placed inside the chamber. Anaerobic conditions are created by adding water to a gas generator envelope that is placed in the jar just before sealing. The envelope contains two chemical tablets, sodium borohydride and sodium bicarbonate.

Water reacts with these chemicals, producing hydrogen gas and carbon dioxide. The hydrogen gas combines with free oxygen in the chamber to produce water, removing all free oxygen from the chamber. This reaction is catalyzed by the element palladium, which is attached to the underside of the lid of the jar. The carbon dioxide replaces the removed oxygen, creating a completely anaerobic environment.

Requirements: 1.Anaerobic container. 2.Anaerobic gas generating kits. 3.Anaerobic indicators.

Technique Technique: Aseptically inoculate the media. Place the inoculated plates into the plate carrier. Prepare the anaerobic indicator by cutting and exposing it. Use forceps to insert it into the plate carrier ( it will ensure anaerobic conditions by change in its color). Cut off the corner of the kit sachet as indicated by the broken line. Add 10 ml of water. Immediately place the sachet upright in the anaerobic jar. Close the lid of the jar according to instructions.

Cooked meat medium which is used to culture Clostridium and Bacteroides species. The anaerobes grow at the bottom of the medium among the meat particles which contain effective reducing substances. Used to grow anaerobes that might be killed by oxygen. Contain ingredients (reducing agents) that chemically combine with oxygen and remove it from the medium. Example: Sodium thioglycolate II. Use of reducing agents in culture media

Culturing in carbon dioxide Aim Carbon dioxide enriched atmosphere is required for the growth of Neisseria gonorrhoeae, Neisseria meningitidis, Brucella species, and Streptococcus pneumoniae.

Methods for obtaining carbon dioxide conditions. I. Commercially carbon dioxide gas-generating systems are available II. Simple ways of providing a carbon dioxide enriched atmosphere: A. Burning candle: Enclose the inoculated plates in an airtight jar with a lighted candle. As the candle burns, the oxygen content is reduced leaving a carbon dioxide content of 3–5%. Important: It is necessary to use a white wax smokeless candle to avoid the release of fumes which may be bactericidal or interfere with the growth of bacteria.

B.Chemical method Carbon dioxide can be generated chemically in a jar by reacting sodium bicarbonate with tartaric acid or citric acid. Generation of carbon dioxide from chemicals: To obtain a 10% carbon dioxide atmosphere in a jar of about 3 liters capacity, mix 0.7 g sodium bicarbonate with 1.7 g tartaric acid (or 2.4 g citric acid). Immediately before closing the jar, moisten the chemicals with water.