pedagogy.sandhya.pptx
•Download as PPTX, PDF•
0 likes•8 views
Anaerobic culture methods
Recommended
More Related Content
Similar to pedagogy.sandhya.pptx
Similar to pedagogy.sandhya.pptx (20)
More from Sandhya Kulkarni
More from Sandhya Kulkarni (8)
Recently uploaded
Recently uploaded (20)
pedagogy.sandhya.pptx
- 1. Culture Methods for Anaerobes Smell that helps you help a patient
- 2. Introduction • What is an anaerobe? • Reason for intolerance? • Mostly endogenous with few exceptions • Examples of some anaerobes – Clinical picture diagnostic – Clinical picture non – diagnostic
- 3. Clues • Infection adjacent to a mucosal surface • Foul-smelling lesion or discharge • Free gas in tissue or discharges • Typical morphology for anaerobes on Gram stain • No growth or routine bacterial culture (“sterile-pus”) • Growth in anaerobic zone of fluid media • Gas, foul-smelling odor in specimen or bacterial culture
- 4. Scope 1. What samples you could culture for anaerobes? What you can not? 2. What media are used? 3. What are the incubation requirements? And how are they accomplished?
- 5. What samples you could culture for anaerobes? Specimen collection = critical step
- 6. Specimen collection Basic rule to avoid normal flora • Samples accepted – Aspirated by needle and syringe closed abscess sealed with cubber cork ..air tight – Blood – CNS abscess/csf/tissue – Culdocentesis – PID – Thoracocentesis – Empyema – Syringe aspiration – Sinusitis – Aseptic deep excisional biopsy - Tissue – Suprapubic percutaneous needle aspiration of Bladder –Urinary tract
- 7. Specimen Rejected • Sputum • Cervical/Vaginal swab • Stool • Ulcer • Voided urine
- 8. Media for Anaerobic culture - solid • Non -selective • Sheep Blood agar with or without growth factors • Selective agars for organisms • Laked Blood Agar containing Kanamycin and Vancomycin • Phenylethyl alcohol agar • CCFA
- 9. Media for Anaerobic culture - liquid • Thioglycollate broth • Robertson’ s cooked meat broth
- 10. Principle of Methods • Producing vacuum • Displacing oxygen with other gases • Absorption of oxygen by – chemical or • Alkaline pyrogallol • Chromium & sulphuric acid – biological methods • Using reducing agents
- 11. Methods - Anaerobiosis • McIntosh Field’s jar • Air pumped out • Hydrogen gas pumped in • Catalyst – Hydrogen + Oxygen • Reduced methylene blue remains colorless / Pseudomonas aeruginosa
- 12. Methods - Anaerobiosis Sodium borohydride - NaBH4 Sodium bicarbonate - NaHCO3 Citric acid - C3H5O(COOH)3 Cobalt chloride - CoCl2 (catalyst) NaBH4 + 2 H2O = NaBO2 + 4 H2↑ C3H5O(COOH)3 + 2 NaHCO3 + [CoCl2] = C3H5O(COONa)3 + 3 CO2 + 3 H2 + [CoCl2] 2 H2 + O2 + [Catalyst] = 2 H2O + [Catalyst]
- 13. Methods - Anaerobiosis GLOVE BOX
- 14. Summary • Sample collection and transport is the most critical phase in isolating anaerobes • Anaerobic jar with gaspak is the routine method • Novel methods of diagnosing anaerobes such as GLC, molecular or immunologic methods are not popular • Hence, these methods are all the more relevant to be understood.
Editor's Notes
- McIntosh Fildes Jar It is one of the physical methods to produce anaerobiasis. It is used to produce anaerobic environment during the incubation of anaerobic cultures in the laboratory. It works on the principle of evacuation and replacement, where the air inside the chamber is evacuated and replaced with hydrogen or a mixture of gases. McIntosh and Fildes jar consists of a stout glass or metal (8x5”) jar with a lid that can be tightly clamped with a screw to make it airtight. The lid has two taps, one acts as outlet that is connected to a vacuum pump and the other as inlet through which hydrogen gas is supplied. The lid also consists of two electrical terminals, which is connected on the underside to a small porcelain spool wrapped by a layer of palladinised asbestos. Subsequent models did not have electrical terminals as cold catalyst was being used. The presence of air is deleterious for many anaerobic bacteria and must be incubated in its absence. The inoculated culture plates are placed inside a metal jar and the lid clamped tight. The air inside is removed using a vacuum pump. The pressure inside the chamber is reduced to 100 mm below mercury. It is not practically possible to evacuate all the air since it will cause boiling in the liquid or detachment of the medium. Despite evacuation of air, some amount of oxygen will still be left behind. Hence the air is replaced with hydrogen gas from a cylinder. For convenience sake, a football bladder filled with hydrogen was used earlier. The pressure inside the chamber is brought back to atmospheric pressure (760mm Hg). Using a palladium catalyst residual oxygen can be made to react with hydrogen to form water, but this reaction is not spontaneous. This reaction is catalysed by palladium catalyst that is heated using electricity. As the reaction continues more hydrogen is used up. This process is allowed to continue for 20 minutes. Use of hydrogen gas and use of electricity can lead to explosions. Hence hydrogen gas has been substituted by a mixture of gases (consisting of 5%CO2, 10%H2 and 85%N2) and a cold catalyst consisting of an alumina tablet coated with palladium. The jar is then placed inside an incubator at 37o C for 48 hours. Efficiency indicator: Efficacy of anaerobiasis produced and maintained inside the jar can be checked using a methylene blue indicator. This indicator is made up of three stock solutions: 6% glucose in distilled water, 6% solution of N/10 NaOH and 3 ml of 0.5% aqueous methylene blue diluted to 100 ml with distilled water. Equal volumes of these three reagents are mixed in a test-tube and boiled. Boiling reduces methylene blue, turning it colorless. Methylene blue is colorless in reduced conditions and turns blue when oxidized. This tube had to kept inside the jar along with the culture plates. Subsequent models had a side arm to which a tube filled with Luca’s semisolid indicator was attached. Disadvantages: Palladium catalyst is inactivated by excess moisture and has to be rejuvenated by heating them at 160o C for two hours. Subsequently, introduction of silica gel absorbent solved this problem. This system is excellent but requires skill to operate and special apparatus that are costly. Requirements of vacuum pump and supply of gas is a major drawback of this system and hence it is being replaced by more convenient GasPak system. The jar, about 20″×12.5″ is made up of a metal. Its parts are as follows: The body made up of metal (airtight) The lid, also metal can be placed in an airtight fashion A screw going through a curved metal strip to secure and hold the lid in place A thermometer to measuring the internal temperature A pressure gauge to measuring the internal pressure (or a side tube is attached to a manometer) Another side tube for evacuation and introduction of gases (to a gas cylinder or a vacuum pump) Method of use[edit] First: The culture: The culture media are placed inside the jar, stacked up one on the other, and Indicator system: Pseudomonas aeruginosa, inoculated on to a nutrient agar plate is kept inside the jar along with the other plates. This bacteria need oxygen to grow (aerobic). A growth free culture plate at the end of the process indicates a successful anaerobiosis. However, P. aeruginosa possesses a denitrification pathway. If nitrate is present in the media, P. aeruginosa may still grow under anaerobic conditions. Second: 6/7ths of the air inside is pumped out and replaced with either unmixed Hydrogen or as a 10%CO2+90%H2 mixture. The catalyst (Palladium) acts and the oxygen is used up in forming water with the hydrogen. The manometer registers this as a fall in the internal pressure of the jar. Third: Hydrogen is pumped in to fill up the jar so that the pressure inside equals atmospheric pressure. The jar is now incubated at desired temperature settings. A wire cage hanging from the lid to hold a catalyst that makes hydrogen react to oxygen without the need of any ignition source
- 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, and 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.