ANAEROBIC CULTURE METHODS.pptx for education

education

1. ANAEROBIC CULTURE METHODS ARYA 1 st MSc MICROBIOLOGY

2. INTRODUCTION Anaerobic bacteria grow only in the absence of oxygen –often extremely toxic. Anaerobic bacteria differ in their requirements of and sensitivity to oxygen. Some such as C.histolyticum are aerotolerant and may produce some growth on the surface of aerobic plates ,while others such as C.tetani are strict anaerobes and form surface growth only if the oxygen tension is less than 2mmHg. Facultative anaerobes:Can grow in the presence/absence of oxygen. Obtain energy by both respiration and fermentation Oxygen not toxic, some use nitrate or sulphate as a terminal electron acceptor under anaerobic conditions.

3. Obligate /strict anaerobes:Oxygen is toxic to these organisms, do not use oxygen as terminal electron acceptor. Microaerophilic organism:Require low level of oxygen for growth ,but cannot tolerate the levels present in the atmosphere. eg : H pylori Aerotolerant anaerobes:Metabolism is anaerobic but they are unaffected by the presence of oxygen OXYGEN TOXIXITY :Oxygen is used by aerobic and facultatively anaerobic organisms as its strong oxidizing ability makes it as excellent electron acceptor During the stepwise reduction of oxygen, which takes place in respiration toxic and highly reactive intermediates are produced reactive oxygen species (ROS).

4. FACTORS THAT INHIBIT THE GROWTH OF ANAEROBES BY OXYGEN 1.Toxic compounds are produced Eg:H2O2 , Superoxides 2.Absence of catalase and superoxide dismutase 3.Oxidation of essential sulfhydryl groups in enzymes without sufficient reducing power to regenerate them

5. CULTURING OF ANAEROBES Culture of anaerobes is extremely difficult due to the need to exclude oxygen,slow growth and complex growth requirements Molecular methods based on DNA analysis and direct microscopy have shown that we are largely ignorent of the microbial world and previously unknown diversity has been discovered

6. ANAEROBIC CULTURE METHODS Production of vacuum Displacement of oxygen Displacement and combustion of oxygen Absorption of oxygen by chemical or biological methods By readucing agents Anaerobic chamber

7. PRODUCTION OF VACUUM This is achieved by incubating cultures in a vacuum dessicator . Is not an effective method . Not in use now DISPLACEMENT OF OXYGEN Bt inert gases like hydrogen, nitrogen, carbon dioxide or helium. A lighted candle is kept in a large air tight container loaded with inoculated plates. It is expected that burning candle will use up all the oxygen inside before it is extinguished but some amount of oxygen is always left behind. Unsatisfactory.

8. Displacement & combustion of oxygen This involves evacuation of air from the jar and replacement with inert gas like hydrogen followed by removal of residual oxygen by use of a catalyst. McIntosh and filde’s anaerobic jar. Anoxomat instrument . McIntosh and filde’s anaerobic jar. This is the most reliable and widely used anerobic metod . Consists of a stout glass or metal jar with a metal lid that can be clamped air tight with a screw. Lid has 2 tubes with taps, one acting as gas inlet and other as outlet. Lid has 2 terminals connected to an electric supply.

9. A catalyst alumina pellets coated with palladium is suspended under the lid by small wires which are connected with the terminals to heat the catalyst for its activity. Now a days catalyst at room temperature is used. Culture plates inoculated with specimens are placed inside the anaerobic jar with an indicator. Tight the lid, outlet tube connected to vacuum pump while inlet tube is closed. Air inside is evacated and outlet tube is closed and hydrogen gas is passed through inlet tube till reduced atmospheric pressure is brought to normal atmospheric pressure(760mmHg) which is monitored on vacuum gauze as zero Electric terminals are switched on to heat the catalyst and this helps to combine hydrogen and residual oxygen to water. Reduced methylene blue is generally used as indicator of anaerobiosis in jar which remains colourless in anaerobic conditions, but turns blue on exposure to oxygen

10. Anoxomat Is the new standard for culturing anaerobes and microaerobes in virtually any laboratory , clinical or industrial. This is achieved according the evaluation – replacement method of Maclntosh & Fildes , by means of a microprocessor controlled automatic valve system. Is an automatic equipment which evacuates the air from jar and replaces by hydrogen gas from a cylinder . Same catalyst is used here to remove the traces of oxygen. More easier. Advantages Is rapid :anaerobic condition in 70 sec and microaerobic in 20 sec Easy to operate Is space saving Is multifunctional Is cost saving Produces no environmental pollution, no chemical waste, no plastics Can be used continously : 365 days no start up time, maintenance and clearing is not necessary Can handle both fluid as well as fixed media.

11. ABSORPTION OF OXYGEN BY CHEMICAL METH ODS PYROGALLOL Alkaline pyrogallol absorbs oxygen. A large tube containing solution of NaOH and pyrogallic acid placed inside air tight jar produces anaerobic conditions. CHROMIUM AND SULPHURIC ACID A mixture of chromium and sulphuric acid is used They two react in presence of oxygen and produce chromous sulphate GAS PACK SYSTEM Most commonly used method Very simple to perform Uses a sachet containing sodium biocarbonate and sodium borohydride which react chemically in presence of water to produce hydrogen and CO2 . Cold catalyst –permits combination of hydrogen and oxygen Reduced methylene blue is used as indicator In anaerobic condition it is colourless and becomes blue on exposure to oxygen

12. Traces of O2 is removed by using same catalyst in McIntosh Fildes method.Anaerobic indicator strips included to monitor the anaerobic condition .

13. BY REDUCING AGENTS Oxygen in culture media can be reduced by various agents such as glucose , thioglycollate ,cooked meat pieces , cystein and ascorbic acid Based on this principle 2 most commonly used liquid culture media are THIOGLYCOLLATE BROTH COOKED MEAT BROTH(RCM)

14. ANAEROBIC CHAMBER Is an anaerobic incubation system Also known as anaerobic glove boxes Provides oxygen free environment Is fitted w ith airtight rubber gloves to insert hands for working with specimens Contains catalyst, dessicant , H gas, CO2, N

15. PRE REDUCED ANAEROBICALLY STERILIZED (PRAS) Are prepared entirely under O2 free condition Media used in the anaerobic bacteriology can be freshly prepared or  purchased from commercial suppliers.   Media for anaerobic culture prepared in the laboratory should be used within 2 weeks of preparation as long storage degrades the quality of the media due to peroxides accumulation and dehydration.  Anaerobic culture media contains reducing agents such as cysteine. Pre-reduced, anaerobically sterilized media are produced bydifferent  commercial suppliers, which have extended shelf life of up to six months.  The primary plating media for inoculating anaerobic specimen includes a non-selective blood agar and one or all of the following mentioned selective media

16. Non selective media used in anaerobic bacteriology: Cooked meat broth (e.g. Robertson’s Cooked Meat Medium): Non-selective for cultivation of anaerobic organisms; with addition of glucose, can be used for gas-liquid chromatography. Anaerobic blood agar : It is a non-selective medium for isolation of anaerobes and facultative anaerobes. Egg-yolk agar (EYA): Non-selective for determination of lecithinase and lipase production by Clostridia and Fusobacteria . Peptone-yeast extract glucose broth (PYG) : Non-selective for cultivation of anaerobic bacteria for gas-liquid chromatography. Selective and differential media used in anaerobic bacteriology : Bacterioides bile esculin agar (BBE) : It is selective and differential for Bacteriodes fragilis group and good for presumptive identification. Laked Kanamycin- vancomycin blood agar (LKV) : It is selective for isolation of Prevotella and Bacteriodes spp. Anaerobic phenylethyl alcohol agar (PEA): Selective for inhibition of gram negative rods and swarming by some Clostridia. Cycloserine cefoxitin fructose agar (CCFA):  selective for Clostridium difficile . Thioglycollate broth : Non selective for cultivation of anaerobes; as well as facultative anaerobes and aerobes.

17. HUNGATE TECHINIQUE Hungate Type Anaerobic Culture Tube Gas tight hungate tubes designed for obtaining and maintaining anaerobic culture conditions. Method of hungate et.al , utilizes syringe methods for anaerobiosis Anaerobic culture hungate tubes contain 3 autoclavable parts a scerw cap with 9mm opening a flange type non toxic rubber stopper that is gas permeable a disposible screw cap culture tube The Hungate anaerobic culture tube was developed for growing and maintaining anaerobic bacteria Technique is simple Exclude o2 by flushing the tube with desired gas Place 4.5ml of pre reduced anaerobic agar medium into tube Seal the tube with the butyl rubber stopper and screw cap Autoclave the tube Inoculate with syringe Prepare on roll tube spinner Incubate in water bath

18. QUALITY ASSURANCE ( Indicator of anaerobiosis ) Chemical indicator: reduced methelene blue remains colourless in anaerobic condition and turns to blue on exposure to O2 Biological indicator: plate inoculated with pseudomonas is incubated along with other inoculated plates for anaerobic culture.Absence of growth indicates perfect anaerobiosis

19. THANK YOU