Culturing bacterial Pathogens

1. Culturing bacterial Pathogens

2. 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 Pseudomonasand • Food borne illnesses, which can be caused by bacteria such as Shigella, Campylobacterand Salmonella.

3. 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.

4. Temperature of incubation • Optimum temperature: The temperature at which a microorganism grows best. • The temperature below which growth stops is called the minimum temperature and that above which growth stops is called the maximum temperature. • The temperature selected for routine culturing is 35–37 0C done inside incubator. • Growth of microorganisms is more affected by slight rises above their optimum temperature than by reductions below it.

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

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

8. 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

9. 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.

10. Presence of O2 and CO2 in incubator According to the gaseous requirements microorganisms are:

11. The Requirements for Growth: Related to Oxygen • Oxygen (O2) Dr.T.V.Rao MD Table 6.1

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

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

14. I. Commercially produced kits TheGasPak 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.

15. 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.

16. Requirements: Anaerobic container. Anaerobic gas generating kits. Anaerobic indicators.

18. 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.

19. II. Use of reducing agents in culture media • Cooked meat medium which is used to culture Clostridium andBacteroides 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

20. Culturing in carbon dioxide Aim • Carbon dioxide enriched atmosphere is required for the growth of Neisseriagonorrhoeae, Neisseriameningitidis, Brucella species, and Streptococcus pneumoniae.

21. 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: • 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.

23. 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.