Anaerobes P3 (laboratory diagnosis)

tissue biopsy or aspiration using needle and syringe

In general, material for anaerobic culture is best obtained by __________ or __________________.

swabs

The use of _____ is a poor alternative because of excessive exposure of specimen to the deleterious effects of drying, the possibility of contamination during collection, and easy retention of microorganisms in the fibers of the swab.

public health laboratory

Specimen collection for Clostridium: Food and freshly passed fecal specimens must be sent to a _______________ for confirmation of C. perfringens food poisoning

4 degrees C

Specimen collection for Clostridium: These should be transported at ___

24

Specimen collection for Clostridium: Processed within ___ hours of collection

botulinum toxin; serum, feces, vomitus, gastric contents

Specimen collection for Clostridium: The clinical diagnosis of botulism is confirmed by demonstration of ___________ in ____, _____, _____, or ___________, as well as by recovery of the organism from the stool of patients.

2 hours

Specimen collection for Clostridium: Specimens should be cultured within ________ after collection

toxin A & toxin B

C. difficile infection (CDI) indicates collection of a freshly passed stool specimen for culture and toxin assays for both ______ and _______

liquid or unformed stools

Only ______________ should be processed for CDI to prevent the treatment of patients colonized with the bacterium.

Formed stools or rectal swabs

_______ or ________ are adequate to detect carriers.

1. Rubber-stoppered collection vial containing an agar indicator system.

2. Vacutainer Anaerobic Specimen Collector

3. Anaerobic Transport System for Tissue Specimens

3 kinds of anaerobe transport system

Rubber-stoppered collection vial containing an agar indicator system

Vial is gassed out with oxygen-free carbon dioxide (CO2) or nitrogen

swab

0. VACUTAINER ANAEROBIC SPECIMEN COLLECTOR

   • If only a ____ specimen can be obtained, a special collection device with an oxygen-free atmosphere is required.

   • When the swab is reinserted, care must be taken not to tip the container, which would cause the oxygen-free CO2 or nitrogen to spill out and be displaced by ambient air.

   • The sterile pack contains a sterile swab and an oxygen- free inner tube.

   • After the specimen has been collected, the swab is inserted back into the inner tube.

   • Agar on the bottom of the outer tube contains an oxygen tension indicator

spill out

0. VACUTAINER ANAEROBIC SPECIMEN COLLECTOR

   • If only a swab specimen can be obtained, a special collection device with an oxygen-free atmosphere is required.

   • When the swab is reinserted, care must be taken not to tip the container, which would cause the oxygen-free CO2 or nitrogen to _____ and be displaced by ambient air.

   • The sterile pack contains a sterile swab and an oxygen- free inner tube.

   • After the specimen has been collected, the swab is inserted back into the inner tube.

   • Agar on the bottom of the outer tube contains an oxygen tension indicator

oxygen- free

0. VACUTAINER ANAEROBIC SPECIMEN COLLECTOR

   • If only a swab specimen can be obtained, a special collection device with an oxygen-free atmosphere is required.

   • When the swab is reinserted, care must be taken not to tip the container, which would cause the oxygen-free CO2 or nitrogen to spill out and be displaced by ambient air.

   • The sterile pack contains a sterile swab and an _________ inner tube.

   • After the specimen has been collected, the swab is inserted back into the inner tube.

   • Agar on the bottom of the outer tube contains an oxygen tension indicator

oxygen tension indicator

0. VACUTAINER ANAEROBIC SPECIMEN COLLECTOR

   • If only a swab specimen can be obtained, a special collection device with an oxygen-free atmosphere is required.

   • When the swab is reinserted, care must be taken not to tip the container, which would cause the oxygen-free CO2 or nitrogen to spill out and be displaced by ambient air.

   • The sterile pack contains a sterile swab and an oxygen-free inner tube.

   • After the specimen has been collected, the swab is inserted back into the inner tube.

   • Agar on the bottom of the outer tube contains an _____________

saline

ANAEROBIC TRANSPORT SYSTEM FOR TISSUE SPECIMENS
Tissue is placed in a small amount of ______ to keep it moist.
It then is inserted into a self-contained, atmosphere- generating anaerobic bag for transportation.

This system is called the GasPak Pouch
It is held at room temperature pending processing in the laboratory, because refrigeration can oxygenate the specimen

self-contained, atmosphere- generating anaerobic bag

ANAEROBIC TRANSPORT SYSTEM FOR TISSUE SPECIMENS
Tissue is placed in a small amount of saline to keep it moist.
It then is inserted into a _____________ for transportation.

This system is called the GasPak Pouch
It is held at room temperature pending processing in the laboratory, because refrigeration can oxygenate the specimen

GasPak Pouch

ANAEROBIC TRANSPORT SYSTEM FOR TISSUE SPECIMENS
Tissue is placed in a small amount of saline to keep it moist.
It then is inserted into a self-contained, atmosphere- generating anaerobic bag for transportation.

This system is called the ___________
It is held at room temperature pending processing in the laboratory, because refrigeration can oxygenate the specimen

room temperature

ANAEROBIC TRANSPORT SYSTEM FOR TISSUE SPECIMENS
Tissue is placed in a small amount of saline to keep it moist.
It then is inserted into a self-contained, atmosphere- generating anaerobic bag for transportation.

This system is called the GasPak Pouch
It is held at _____________ pending processing in the laboratory, because refrigeration can oxygenate the specimen

Gram staining

important rapid tool for anaerobic bacteriology

Gram staining

reveal the types and relative numbers of microorganisms and host cells present

Gram staining

Serves as a quality control measure for the adequacy of anaerobic techniques

Gram staining

You are first going to see through this your specimen for presence of organism. It will give you an idea whether the possible organism that can grow in your culture are gram-positive rods/cocci, or gram- negative rods/cocci.

1. Poor transport methods

2. Excessive exposure to air during specimen processing

3. Failure of the system (jar, pouch, or chamber) to achieve an anaerobic atmosphere

4. Inadequate types of media or old media

5. Killing of microorganisms by antimicrobial therapy

A positive Gram stain with a negative culture may indicate:

anaerobic chamber

Specimen processing
- Once you received the specimen, you are going to process it inside an _____________

0% oxygen

Specimen collection

- Black parts for hands in order to maintain __________ environment.

- This is a gloveless anaerobe chamber

gloveless

Specimen processing
- Once you received the specimen, you are going to process it inside an anaerobic chamber

- Black parts for hands in order to maintain 0% oxygen environment.

- This is a __________ anaerobe chamber

GasPak Anaerobic System

It is where inoculated culture media is placed

1. Polycarbonate jar

2. Lid fitted with gasket

3. Indicator strip

4. Indicator

5. Disposable gas generator pouch

6. Palladium catalyst pallets

7. GasPak Jar

8. CO₂

9. Methylene blue

10. Resazurin

components of GasPak Anaerobic system

Indicator strip

A strip that has been impregnated with indicator of oxygen-reduction like methylene blue or resazurin

Indicator

It is used when GasPak is exposed to oxygen.
Helps in determining when anaerobic environment is maintained inside the GasPak.

Disposable gas generator pouch

It contains sodium bicarbonate and sodium borohydride

Palladium catalyst pallets

Catalyse the reaction to remove oxygen

GasPak Jar

Use reagents that simply require the addition of water to catalyze a reaction

CO2

Is used to replace the oxygen removed, resulting in anaerobic atmosphere.

Colorless; Blue

Methylene blue
_______: in anaerobic environments
______: in aerobic environments

Colorless/White

PINK

Resazurin
A redox indicator
________: in anaerobic environments
_____: in aerobic environments

Principle of GasPak anaerobic system

- Tubes or plates that have been inoculated are placed inside the polycarbonate container or anaerobic jar together with gas generator sachet as well as an indicator strip and sealed when water is present and the chemicals contained in the bag (sodium bicarbonate and sodium thermohydride) interact chemically and produce carbon dioxide and hydrogen gas.

sodium bicarbonate and sodium thermohydride

Principle of GasPak anaerobic system

- Tubes or plates that have been inoculated are placed inside the polycarbonate container or anaerobic jar together with gas generator sachet as well as an indicator strip and sealed when water is present and the chemicals contained in the bag (___________ and _________) interact chemically and produce carbon dioxide and hydrogen gas.

carbon dioxide and hydrogen gas

Principle of GasPak anaerobic system
- Tubes or plates that have been inoculated are placed inside the polycarbonate container or anaerobic jar together with gas generator sachet as well as an indicator strip and sealed when water is present and the chemicals contained in the bag (sodium bicarbonate and sodium thermohydride) interact chemically and produce ________ and ________

condensation; palladium mineral

PRINCIPLE OF GASPAK ANAEROBIC SYSTEM

The hydrogen resulting from the reaction reacts with the oxygen inside the jar and creates water which is formed as ________ in the interior of the container

This reaction is triggered by the _____________ that is affixed to the underside of the lid of the container.

Carbon dioxide

PRINCIPLE OF GASPAK ANAEROBIC SYSTEM

____________ is used to replace the oxygen removed resulting in an anaerobic atmosphere.

mercury

nitrogen

Evacuation replacement method

- air is removed from sealed jar by drawing a vacuum of 25 inches (62.5cm) of _____

- The process is repeated two times with the jar being filled with an oxygen-free gas, such as _______ between evacuation.

• The final fill of the jar is made with a gas mixture.

• Obligate Anaerobes should grow in a 0% oxygen environment.

80-90%

5-10%

5-10%

Evacuation replacement method

components for gas mixture

nitrogen =

hydrogen =

carbon dioxide =

CO2

many anaerobes require ____ for maximal growth