I&I Week 5 Veterinary Bacteriology: Sample collection, culture, and diagnosis

What are some important considerations when collecting bacteriological samples for the lab?

Aseptic collection, sampling early following onset of clinical signs, avoiding contamination, and informing the lab if treatment has started

What types of samples are suitable for anaerobic culture?

Normally sterile body fluids, surgical specimens from sterile sites, deep abscess contents, aspirates from deep wounds, and blood collected aseptically

What specimens are unsuitable for anaerobic culture?

Saliva, vaginal or cervical specimens, feces (unless looking for a particular clostridial pathogen), tracheal swabs, naso-tracheal aspirates, colostomy or ileostomy effluents, skin or superficial wound swabs, voided or catheterized urine

What clinical conditions suggest anaerobic infections?

Foul-smelling discharges, deep infections, necrotic tissue, gas in tissue, endocarditis with negative aerobic blood culture

What are some techniques used in the direct examination of bacteriological specimens?

Macroscopic observations, staining (Gram, Dilute carbol fuschin, Ziehl Neelsen), and microscopy

What information can be gathered from microscopic examination of stained bacteriological specimens?

Bacterial morphology, size, shape, arrangement, staining affinity, spore formation, and capsule formation

Name some indirect methods used to identify bacteria?

Immunology and Serology, ELISA, Agglutination tests, Precipitation, Complement fixation, Immunomagnetic separation, and Fluorescent Antibody Testing

What are the advantages of using immunological and serological methods for bacterial identification?

Detection limit for organisms/antigens with low abundance, difficulties in generating selective antibodies

What are some limitations of bacterial culture for diagnosis?

Can take a long time for results, contamination is possible, and requires the right sample at the right time

What are the advantages of using PCR in diagnosing bacterial infections?

Rapid, requires a small amount of sample, and can detect fastidious, in-culturable, slow-growing, or dangerous pathogens, with high specificity and sensitivity

What are the disadvantages of using PCR in diagnosing bacterial infections?

Equipment may be expensive, and primers vary in specificity

What are the applications of PCR?

Research, diagnostics, and sequencing

What are the advantages of whole genome sequencing?

Provides the most detail and unambiguous information

What are the disadvantages of whole genome sequencing?

Expensive and has a detection limit for organisms with low abundance

What are the advantages of using MALDI-TOF MS for diagnosing bacterial infections?

High-throughput, rapid results, generates easily interpretable spectra, qualitative and quantitative data, and low overall operating costs

What are the disadvantages of using MALDI-TOF MS for diagnosing bacterial infections?

Detection limit for organisms with low abundance, host proteins and normal flora might overlap mass spectra, high initial investments and maintenance costs, and lacking differentiation of closely related species

What are the requirements for collecting tissues and organs for bacterial isolation?

Use sterile instruments, collect at least 1cm3 of sample, place in individual polyethylene or sterile screw-capped jars

When should postmortem material be collected for bacterial isolation?

As soon as possible after death

What samples should be collected in cases of abortion?

The whole fetus or pieces of the fetus, pieces of tissue, abomasal contents (ruminants), and uterine discharge

Why are fluids preferred over swabs for bacterial isolation?

The greater the volume, the greater the likelihood of detecting the causal organism

Why are short cotton swabs unsatisfactory for nasopharyngeal specimens?

They do not collect enough epithelial cells and mucus

When are guarded swabs necessary?

For certain bacteriological examinations where contamination by normal flora may pose a problem

What are the preferred ways of submitting specimens from nasal passages, pharynx, tonsil, eye, ear, vagina, and cervix?

Swabs and discharges

How should feces be collected for bacterial isolation?

Directly from the rectum in a manner to avoid contamination

How much feces should be forwarded to the lab without transport medium?

A sample about the size of the end of the thumb

Why should fecal swabs be placed in medium?

To avoid desiccation

How should milk samples be collected from individual cows?

As soon as mastitis is observed, in sterile vials or tubes, after cleaning the udders

When should milk not be collected for bacterial isolation?

Not after treatment

How should the udder be prepared for aseptic milk collection?

Wipe teats vigorously using 70% ethyl alcohol on cotton wool, paying special attention to the teat sphincters. If washed, dry thoroughly with a paper towel

How should the sterile collecting bottle be held during milk collection?

Almost horizontally

What should be done with the first milk from each teat?

Discard it

How should a composite milk sample be collected?

Take a little milk from each teat, collecting milk from the nearest teats first

How should eye samples be collected for bacterial isolation?

A conjunctival swab may be taken or scrapings can be taken with a fine, sterile spatula and washed into transport media

How should urine be collected for bacterial microscopy and culture?

By cystocentesis, catheter, or midstream urine

How should abscess samples be collected for bacterial isolation?

Collect 3 ml of pus with scrapings from the wall of the abscess

Why should pus be collected from the wall of the abscess?

Pus at the center of the abscess is often sterile

Which abscesses yield the best cultural results?

Freshly formed abscesses

What type of bacteria can often be cultured from abscesses?

Anaerobic bacteria

How should skin lesions be sampled for bacterial isolation if intact pustules or vesicles are present?

Disinfect the surface with 70% ethyl alcohol, allow to dry, and aspirate material with a sterile syringe and fine needle

How should skin lesions be sampled if there is a raw surface of the ulcer?

A swab may be taken from the raw surface of the ulcer

How should a biopsy of a wound be collected?

After the superficial area has been cleaned and debrided

How should samples be collected when dermatophytosis/ringworm is suspected?

Hair should be plucked from the lesion and the edge of the lesion should be scraped with a blunt scalpel until blood begins to ooze. The plucked hair and skin scrapings (with the scalpel) and scab material should be submitted

What is the first step in identifying aerobic Gram-positive cocci?

Catalase test

What does it mean if aerobic Gram-positive cocci are catalase-positive?

They are Staphylococci

What is the next step after determining that aerobic Gram-positive cocci are Staphylococci?

Coagulase test

What are some examples of coagulase-positive Staphylococci?

S. aureus, S. pseudintermedius, S. hyicus

What does it mean if aerobic Gram-positive cocci are catalase-negative?

They are Streptococci

What is the next step after determining that aerobic Gram-positive cocci are Streptococci?

Observation of types of hemolysis

Name some spore-forming aerobic Gram-positive rods?

Bacillus spp

Name some non-spore-forming aerobic Gram-positive rods that are catalase-positive and motile?

Listeria spp

Name some non-spore-forming aerobic Gram-positive rods that are catalase-positive and non-motile?

Corynebacterium spp., Rhodococcus equi

Name a catalase-negative, beta-hemolytic, non-spore-forming aerobic Gram-positive rod?

Trueperella pyogenes

Name a catalase-negative, alpha-hemolytic, non-spore-forming aerobic Gram-positive rod?

Erysipelothrix rhusiopathiae

What characteristic is used to differentiate aerobic Gram-negative rods?

Oxidase test

What is the doubling time of E. coli?

20 minutes

What is the doubling time of Mycobacterium tuberculosis?

24 hours

What factors influence the growth of bacteria in culture?

Temperature, hydrogen ion concentration, availability of moisture, atmospheric composition, and osmotic pressure

What are the types of media based on consistency?

Broth (liquid), semi-solid, and solid

How do bacteria grow in liquid media?

Uniformly, producing turbidity

What is nutrient broth?

A general purpose liquid media that grows non-fastidious bacteria

What is the agar content in semi-solid media?

0.2-0.5%

What is the function of semi-solid media?

To demonstrate bacterial motility

What is the most common solidifying agent added to liquid media to make it solid?

Agar

What is a basal media?

A media that supports most non-fastidious bacteria

What is an example of a basal media?

Peptone water

What are enrichment media?

Media with extra nutrients added, such as blood or serum

What are some examples of enriched media?

Blood agar and chocolate agar

What are selective media?

Media that inhibits unwanted commensal or contaminating bacteria to help recover a pathogen from a mixture

How can media be made selective?

By adding antibiotics, dyes, chemicals, or altering the pH

What are differential or indicator media?

Media where different bacteria can be recognized based on their colony morphology

How do differential or indicator media work?

By incorporation of dyes or metabolic substrates

What are some examples of differential media?

MacConkey agar and XLD agar

What is the purpose of Mueller Hinton Agar?

Antimicrobial sensitivity testing

What are the responsibilities of the clinician in ensuring successful bacteriology results?

To collect and submit the appropriate samples accompanied by specific requests or adequate history