Cultivation of Anaerobic Bacteria

Presentation about intrduction and types of anaerobic bacteria and about various methods used for cultivation of anaerobic bacteria

1. CULTIVATION OF ANAEROBIC BACTERIA Mr. Krishnakant B. Bhelkar Asst. Professor, Gurunanak College of Pharmacy, Nagpur Unit I Pharmaceutical Microbiology B. Pharm III Semester

2. ANAEROBIC BACTERIA An anaerobic bacteria  or anaerobe is any organism that does not require oxygen for growth. It may react negatively or even die if free oxygen is present. Depending on amount of oxygen tolerated, they are divided in three types Obligate Anaerobe Aero-Tolerant Anaerobes Facultative Anaerobes

3. ANAEROBIC BACTERIA Depending on amount of oxygen tolerated, they are divided in three types Obligate Anaerobe These are microorganisms killed by normal atmospheric concentrations of oxygen (20.95% O 2 ). Oxygen tolerance varies between species, Some capable of surviving in up to 8% oxygen, others losing viability unless the oxygen concentration is less than 0.5% Aero-Tolerant A naerobes Survive in presence of oxygen – Do not use oxygen for energy requirements but use  fermentation  to produce  ATP. They do not utilize oxygen, They can protect themselves from reactive oxygen molecules.  Facultative Anaerobes A  facultative anaerobe is an organism that makes ATP by  aerobic respiration  if oxygen is present, but is capable of switching to  fermentation if oxygen is absent.

4. METHODS FOR CULTIVATION OF ANAEROBIC BACTERIA Candle Jar Method Anaerobic Jar Method Anaerobic Chamber Vacuum and Gas Displacement Method Thioglycollate Broth Method Alkalline – Pyrogallol Method Brewer Anaerobic Culture Plate Method

5. CANDLE JAR METHOD In this method, media plates with bacterial inoculum is kept inside the jar. Seal the jar with the lit candle inside. The candle flame will consume most of the oxygen in the jar and will produce an elevated level of carbon dioxide. These conditions are ideal for the growth of anaerobic and microaerophilic organisms.

6. CANDLE JAR METHOD

7. ANAEROBIC JAR METHOD In this method, media plates with bacterial inoculum is kept inside the jar. The jar contains palladium pellets, that produces hydrogen inside the jar. The hydrogen reacts with oxygen present inside the jar and form water molecules. This cause reduction in oxygen concentration inside the jar. The anaerobic condition can be confirmed by indicator, methylene blue. Indicator becomes colorless in absence of oxygen This condition is ideal for the growth of anaerobic organisms.

8. ANAEROBIC JAR METHOD

9. ANAEROBIC CHAMBER Anaerobic chambers, also known as anaerobic glove boxes, are atmosphere control units designed to be used when working with oxygen sensitive materials. The chamber contains the sealed environment of H 2 , CO 2 or N 2 . The media plates are kept in the chamber and then N 2 is purged in =side the chamber

10. ANAEROBIC CHAMBER

11. VACUUM AND GAS DISPLACEMENT METHOD Anaerobic In this method, the air in the chamber is removed with the help of applying vacuum and is replaced with the mixture of N2 and CO2, This provides the good condition for cultivation of strict anaerobes.

12. THIOGLYCOLLATE BROTH METHOD Thioglycollate Broth  is a multipurpose, enriched, differential medium used primarily to determine the oxyen requirements of microorganisms. Sodium thioglycolate in the medium consumes oxygen and permits the growth of obligate anaerobes

13. ALKALLINE – PYROGALLOL METHOD Alkaline solutions of  pyrogallol  absorb oxygen efficiently and are used in determining the oxygen content of gas mixtures

14. BREWER ANAEROBIC CULTURE PLATE METHOD Brewer Anaerobic Agar is used for cultivating anaerobic and microaerophilic  bacteria Brewer1 described a special Petri dish cover that allowed surface growth of anaerobes and microaerophiles without anaerobic equipment. A small amount of air is caught over the surface of the medium, and the oxygen in this space reacts with the reducing agents to form an anaerobic environment.

15. BREWER ANAEROBIC CULTURE PLATE METHOD Brewer Anaerobic Agar Approximate Formula* Per Liter Pancreatic Digest of Casein .......................... 5.0 g Proteose Peptone No. 3............................. 10.0 g Yeast Extract .............................................. 5.0 g Dextrose .......................................... 10.0 g Sodium Chloride .................................. 5.0 g Agar ............................................... 20.0 g Sodium Thioglycollate ................................ 2.0 g Sodium Formaldehyde Sulfoxylate ................ 1.0 g Resazurin .............................................. 2.0 mg