BIOL 2010 Lecture 13 - Anaerobic Methods

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Questions and Answers

What should be considered when selecting anaerobic broth for bacterial growth?

It supports growth of almost all bacteria types but is best used with other media. (correct)

It is ideal to use as the sole media for sensitive anaerobes.

It provides inadequate oxygen for facultative anaerobes.

It supports anaerobic growth only.

Which characteristic is NOT an indication of the presence of anaerobes in cultures?

Good growth on aerobic culture plates. (correct)

Brick-red fluorescence on specific agar.

Foul odor when opening an anaerobic container.

Unique morphology on Gram stain of isolates.

Which of the following methods is primarily used to determine if a microorganism is a strict anaerobe or a facultative anaerobe?

Utilization of selective media for Gram-negative anaerobes.

Aerotolerance testing using chocolate agar and anaerobic blood agar. (correct)

Fluorescence examination under UV light.

Colony morphology assessment on aerobic plates.

Which Gram-negative anaerobe is indicated by a double zone of hemolysis on blood agar?

Fusobacterium nucleatum. Signup and view all the answers

What is the expected result of a positive catalase test when performed on an anaerobic organism?

Bubbling upon the addition of hydrogen peroxide. Signup and view all the answers

What is a key characteristic of lecithinase production in cultures on egg yolk agar?

Forms an opaque zone around colonies Signup and view all the answers

Which of the following biochemical techniques is NOT typically used for definitive identification of anaerobes?

Spot Indole testing Signup and view all the answers

Which characteristic describes the proteolytic reaction of certain anaerobic organisms?

Complete and clear zone around colonies Signup and view all the answers

What is a primary function of anaerobic chambers in microbiology?

To allow handling and manipulation of anaerobic cultures Signup and view all the answers

Which method would likely yield definitive identification of anaerobic bacteria?

Cellular fatty acid analysis by gas-liquid chromatography Signup and view all the answers

What observation is typical after testing for lipase activity in cultures on egg yolk agar?

Iridescent, multicolored sheen on the agar surface Signup and view all the answers

Which discs would be appropriate for testing anaerobic gram-positive cocci?

SPS and Nitrate discs Signup and view all the answers

Which statement accurately defines an aerotolerance test methodology?

Inoculate both a CHOC culture and a Brucella culture under anaerobic and aerobic conditions Signup and view all the answers

Which component is essential for culturing anaerobes in a laboratory setting?

Hemin Signup and view all the answers

What specific characteristic should be examined in a gram-negative anaerobe during culture identification?

Morphology observed in Gram stain Signup and view all the answers

Which of the following is NOT a consideration when performing presumptive identification of anaerobes?

Presence of aerobic bacteria Signup and view all the answers

What is one of the key advantages of using direct microscopic examination in anaerobic identification?

It helps establish if the infection is polymicrobial or monomicrobial. Signup and view all the answers

Which type of agar is considered the best for recovering anaerobic bacteria in cultures?

CDC blood agar Signup and view all the answers

During an aerotolerance test, what result indicates that an organism is an obligate anaerobe?

No growth in aerobic conditions but growth in anaerobic conditions Signup and view all the answers

Which of the following methods is primarily used for the presumptive identification of anaerobes?

Direct microscopic examination Signup and view all the answers

What does the presence of tiny gram-negative cocci that are gram-variable suggest in anaerobic identification?

Veillonella Signup and view all the answers

Study Notes

Lecture 13 - Anaerobic Methods

Lecture for BIOL 2010
References are Bailey and Scott's (Chapter 40) and Diagnostic Microbiology (Chapter 22)
This lecture discusses anaerobic microorganisms, their characteristics, pathogenesis, and bacterial virulence. It also covers methods for collecting and processing specimens for anaerobic bacteriology.

Anaerobes Defined

Anaerobes can replicate in the absence of oxygen.
Ambient air contains approximately 21% oxygen and 0.04% carbon dioxide.
Obligate anaerobes are killed in the presence of oxygen, and oxygen free radicals lead to immediate cell destruction.
Obligate anaerobes require low oxidation-reduction (redox) potential.
Capnophilic anaerobes require increased carbon dioxide (CO2) levels (5%-10%), and reduced oxygen (O2) to 15%.
Microaerophilic anaerobes require oxygen reduced to 5% or less.
Facultative anaerobes prefer oxygen as an electron acceptor but can grow without oxygen.
Aerotolerant anaerobes tolerate oxygen but do not use it in metabolism. These can be considered moderate anaerobes which are bacteriostatic if exposed to oxygen.

Why Some Organisms Are Anaerobes

Molecular oxygen has a direct toxic effect on some anaerobes. They lack enzymes to break down toxic products.
Toxic products include superoxide anion, hydrogen peroxide, and hydroxyl radical.
Strict aerobic and facultative anaerobic bacteria possess enzymes to break down toxic products, such as superoxide dismutase and catalase.

Where Anaerobes Are Found

Exogenous anaerobes exist outside the bodies of animals. Infections develop when the agent is ingested or enters the body through trauma (e.g. Clostridium, Sarcina, Fusobacterium).
Endogenous anaerobes exist inside animal bodies and become sources of infection (e.g. Bacteroides, Porphyromonas, Prevotella, Fusobacterium, Clostridium, Propionibacterium, anaerobic cocci).

Important Concepts

Proper collection technique for anaerobic samples is crucial to prevent misleading and non-useful clinical results. Sites of infection must be correctly identified.
Rapid processing of specimens is necessary due to oxygen toxicity and specimen drying.

Specimens for Anaerobic Bacteriology

Acceptable specimens include Cerebrospinal fluid (CSF), abscesses, aspirates, surgical specimens, body fluids, and biopsies.
Unacceptable specimens include sputum, swabs of surface organs (e.g., throat, gingival, skin, perirectal), urine (midstream), large bowel content (e.g., stool, colostomy bags), and vaginal swabs.
Ideal specimen collection methodology utilizes needles and syringes to prevent contamination and avoid air exposure.
Specimens should be placed into pre-reduced anaerobically sterilized (PRAS) media immediately after collection to avoid oxygen exposure.

PRAS Transport Medium

PRAS stands for pre-reduced anaerobically sterilized media
This is the ideal medium for transporting samples.
Two methods are acceptable, the tube and the eSwab system.

Processing of Clinical Samples

Plates with inoculated anaerobic cultures require incubation at 35-37 degrees Celsius in anaerobic conditions, and must not be kept out of the anaerobic environment longer than 1 hour.
Anaerobic incubation systems utilize a combination of catalyst (palladium pellets to remove residual oxygen), desiccant (absorbs formed water), and anaerobic gas (mixture of H2, CO2, and Nitrogen).
Quality control for anaerobic systems should be in place including checking physical reactions (e.g. condensation), chemical indicators (e.g., methylene blue or resazurin strips), and biological indicators (e.g., obligate aerobe and anaerobes). The indicators should show changes after proper incubation timing.

Identification of Anaerobes

The identification method of anaerobes depend on the size and capabilities of the laboratory, the clinical site(e.g., sterile site of infection), and the type of the infection.
Presumptive identification is commonly used in most laboratories; it is quick, cost effective, and typically sufficient for treatment. Presumptive identification relies on observations of culture characteristics (color, smell) and Gram stain.
Definitive ID testing is complex and expensive and often unnecessary for treatment.

Presumptive Identification of Anaerobes

Cultural characteristics, aerotolerance tests, Gram stain observation, and quick biochemical tests (including catalase and spot indole tests) are used.
Direct microscopic examination (Gram stain preparation) can help in ruling out or in identifying pathogens, establishing microbial count, and in selection of suitable culture media.

Presumptive ID - Culture

Choice of anaerobic media is vital for accurate recovery.
Anaerobes have special nutritional requirements which include vitamins (vitamin K and Hemin), yeast extract, and nutrient rich media (e.g., CDC blood agar).
Specimens submitted for anaerobic culture often include requests for both aerobic and anaerobic cultures.
Additional medias are required, including: chocolate agar, Brucella agar, Kanamycin-Vancomycin Laked blood agar (KVLB), Phenylethyl Alcohol Agar, Colistin Nalidixic acid Agar, and anaerobic broths (e.g. cooked meat, thioglycollate).

Media for Anaerobes

Several media types are suitable for anaerobic growth including anaerobic blood agar (SBA), Brucella Blood Agar (BA), Kanamycin-Vancomycin Laked Blood Agar (KVLB), and other specialized medias.

Anaerobic Chamber, Jar, and Pouch

Anaerobic chambers, jars, and pouches create anaerobic conditions.
Water-generating systems for jars require a catalyst and water, while waterless systems do not.

Definitive ID Techniques

Multitest systems, cellular fatty acid analysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and 16S ribosomal RNA gene sequencing are definitive identification methods.

Specialized Potency Antimicrobial Disks

These disks are used to confirm Gram stain reactions.
These are NOT used for susceptibility testing. The purpose of the test is to rapidly differentiate types of Bacteria.

Other Special Potency Disks

Nitrate disks test the ability to reduce nitrate.
Sodium polyanethol sulfonate (SPS) disks test for susceptability within peptostreptococcus and peptoniphilus.
Bile disks test for growth in bile.

Lecithinase and Lipase Reactions

Lecithinase and lipase reactions can identify certain microorganisms including species such asC. perfringens and F. necrophorum.

Proteolytic Reactions

Proteolytic reactions are used to identify organisms that produce proteolytic enzymes by observing a clear zone around their colonies . A strong light source can be used to facilitate observations.

Identifying Clues from Culture

Foul odor, characteristic colony morphology, unique morphology on Gram stain, growth on specific media (e.g., BBE), double zone hemolysis on blood agar, and brick-red fluorescence on KVLB agar can point toward anaerobic presence.

Aerotolerance Testing

Aerotolerance tests are used to determine if isolated microorganisms are strict or facultative anaerobes. Plates are inoculated and incubated to observe differences in bacterial growth with and without oxygen.

Summary of Presumptive ID

Presumptions are based on Gram stain and colony morphology on different medias.
Several simple, inexpensive, and rapid procedures are used.
Reviewing these results can provide a presumptive identification.
Presumptive identification is acceptable for treatment of anaerobes.

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Description

This quiz covers Lecture 13 of BIOL 2010, focusing on anaerobic microorganisms and their unique characteristics. Learn about the classifications of anaerobes, their pathogenesis, and methods for specimen collection and processing in anaerobic bacteriology. Explore the vital aspects of anaerobic methods as outlined in Bailey and Scott's and Diagnostic Microbiology texts.