Bacteriology
- Introduction
- Standard containers and equipment
- Collection of specimens
- Storage and despatch of samples
- Summary of available bacteriology tests
Introduction
A bacterial disease can only be diagnosed when a pathogenic organism can be demonstrated, either on smear, culture, using molecular techniques or in tissue sections, in association with pathological changes. Many pathogenic organisms are present in normal animals, e.g. Clostridium perfringens in intestinal contents, so that recovery of the organism alone may not necessarily be significant. In other cases e.g. E. coli, there are many serotypes, but only a few are commonly pathogenic.
Standard containers and equipment
Sterile plastic bottles and jars
These are available commercially in a range of sizes from about 10 to 100 ml. They are used for fluids, discharges, small lesions, etc.
Plastic bags
For larger specimens of. organs or foetuses. The primary receptacle must be leakproof and not contain more than 500g. Plastic bags may be suitable. A minimum bag thickness of 0.1 mm should be used. All bags should be securely sealed by ziplocking or double folding and application of stout rubber bands or equivalent, the bag should be packed inside a second bag which also contains sufficient absorbent material to absorb fluid should the inner bag leak and securely sealed as above. Plastic bags must not be used if bone, horns or other sharp objects may puncture the bag.
Swabs
Swab transport systems for the recovery of organisms are available commercially. They consist of plain cotton wool sterile swabs, which are inserted into a semisolid transport medium (such as Stuart Transport Medium or Amies Charcoal Transport Medium) particularly for fastidious bacteria. before submission to the laboratory. There are different transport mediums available commercially, such as swab/transport medium packs for general purposes, swab/charcoal transport medium for more sensitive organisms, swab/transport systems modified for anaerobe recovery. Some testing involving special stains or antigen testing may require dry swabs or specific transport conditions.
Microscope slides
Use slides of standard size and ensure they are clean. Some commercially available slides are not washed and are not suitable for making satisfactory smears. Slides with frosted ends are preferred for labelling purposes. Ensure slides are packed such that they don't break in transit eg slide holder, wrapped in tissue/paper with sufficient padding.
Milk sample bottles
Sterile 30 ml Macartney bottles or any equivalent sterile, wide-mouthed screw-topped plastic container. e.g. sterile 30 ml universal containers.
Pipettes for collecting vaginal mucus
Polystyrene artificial insemination pipettes, approximately 50 cm long x 2mm internal diameter and 2 ml capacity. These are available commercially.
Pipettes for collecting preputial scrapings from bulls
See Collection of samples from bulls for culture of Campylobacter fetus and Tritrichmonas foetus.
Plastic pipettes made from polystyrene or polypropylene with suggested dimensions of external diameter 9.5 mm, internal diameter 6.4 mm, 61.0 cm in length, straight except for a bend at the end that is held by the operator during collection; the other end, which contacts the preputial surfaces, is bevelled. They should be fitted to 90 ml firm rubber bulbs for suction while scraping.
NB Do not use non-sterile containers, gloves or fragile containers when submitting material for bacteriological examination. Aspirates submitted in syringes with needles will not be examined.
Collection of specimens
Specimens should be collected aseptically and submitted promptly in individual sterile non-leaking, screw-topped, or plastic jars.
If small, the entire lesion or organ should be submitted. If the lesions are large or widespread, submit a portion of the affected tissue containing the lesion and surrounding area. Alternatively an aspirate of the lesion can be taken and transferred to a small sterile container and submitted.
If a septicaemia/bacteraemia is suspected, submit portions of liver, spleen, heart blood and lung, in separate containers.
Swabs
Are generally inferior to the above. In the case of exudates, a smear(s) should be made and submitted with the swab. for culture should be submitted as soon as possible in transort medium (such as Stuart Transport Medium or Amies Charcoal Transport Medium) is preferred., Stuart Transport Medium is unsuitable for contagious equine metritis (Taylorella equigenitalis) transport if the swabs are to be processed more than 24 hours after collection.
Aborted foetuses and foetal membranes
For foetuses from smaller animals (e.g. sheep, goats, pigs) and smaller foetuses from the larger animals, the entire foetus together with the foetal membranes should be enclosed in separate plastic bags and submitted chilled. . are generally inferior to the above. In the case of exudates, a smear(s) should be made and submitted with the swab.
In the case of the large foetuses which cannot be delivered to a laboratory, a post-mortem examination should be conducted and the appropriate specimens should be collected aseptically into sterile jars (see Abortion).
Impression smears of foetal membranes should be taken prior to despatch of specimens.
Portions of the placenta or cotyledons should also be taken and fixed in buffered formalin prior to despatch.
Faecal samples and rectal swabs
Collect approximately 30 g faeces direct from the rectum into a sterile, wide-mothed non-leaking container. Do not overfill the container. (preferred collection method) Rectal swabs, heavily impregnated with faeces, should be submitted in transport medium such as Stuart Transport Medium or Amies Charcoal Transport Medium.
For Johne's faecal culture, see Johne's disease.
NB
If a parasitological examination is also required, a duplicate sample should be collected.
Smears of intestinal mucosa and pathological lesions
Firmly smear suspect area with slide. A number of smears can often be made on each slide. Air dry and wrap separately or place in slide protective holders for transport (commercially available).
Always leave one (frosted) end of the slide clean for handling and labelling. Smears should be clearly labelled.
Intestinal contents
Should be expressed into sterile, widemouthed, non-leaking containers, avoiding gross contamination of the sample or the container(preferred collection method). Swabs of intestinal contents should be placed in a transport medium (such as Stuart Transport Medium or Amies Charcoal Transport Medium).
Milk samples
Wash the udder and the teats thoroughly and dry with a paper towel. Swab the teats with 70 per cent alcohol. The first couple of squirts should be discarded unless used for a field test and, about 10 ml milk squirted into a sterile 30 ml Macartney bottle or sterile 30 ml plastic universal container held nearly horizontally.
Avoid touching the mouth of the bottle with the teat. Code the samples numerically.
The samples must be submitted chilled using either crushed ice or cooling bricks in an insulated container. Milks must be refrigerated if transport is slightly delayed, or frozen if transport is delayed more than 2-3 days.
Preputial material from bulls
Preputial scrapings submitted unchilled in selective transport medium for T foetus. (InPouch TF). See Collection of samples from bulls for culture of Campylobacter fetus and Tritrichomonas foetus
Selective transport medium for T foetus (inPouch TF) together with instructions for the collection and despatch of specimens will be forwarded from the laboratory upon request. InPouch TF
Preputial samples should not be frozen or refrigerated. Keep at temperatures between 18°C and 30°C.
Semen samples
Must be collected aseptically avoiding contamination by preputial material. Samples collected using an artificial vagina are often grossly contaminated unless they are cultured shortly after collection.
From 0.5 to 1.0 ml is sufficient for bacteriological examination. Attempts to collect large volumes by repeated ejaculation increase the risk of contamination.
Urine samples
For routine cultural examinations to be meaningful, urine samples should reach the laboratory within a few hours of collection. If there are likely delays before or during transport, specimens must be chilled, but should be processed by the laboratory within 72 hours of collection.
When direct examination for leptospires is required, submit a fresh urine specimen specifically for leptospiras motility examination at the laboratory within 20 minutes of collection. Otherwise, for leptospire morphology submit a separate urine sample of at least 20 ml, preserved with 0.25 ml of undiluted formalin or 1.5 ml of 10% formalin.
Vaginal mucus samples
Mucus can be collected from the ventral fornix of the vagina and the external os of the cervix by guiding a plastic artificial insemination pipette by hand (per rectum) and applying gentle suction to the external end.
To diagnose bovine venereal campylobacteriosis (BVC) in abortion or infertility cases:
- (Preferred method): Vaginal mucus samples are collected for demonstration of C. fetus subsp venerealis antibodies by BVC ELISA test. PBST diluent and instructions for collection of mucus samples are available from the laboratory. See Diagnosis of bovine venereal campylobacteriosis by ELISA. Vaginal mucus is collected using a plain, sterile cotton swab and placing the cotton swab end into phosphate buffered saline plus Tween (PBST) for transport. The swabs, PBST and instructions are provided by the laboratory.
- For isolation of Campylobacter, direct inoculation to Campylobacter transport media (CTM) is ideal.
If specimens for Campylobacter cuture will not reach the laboratory within 6-8 hours, then sealed pipettes from which air bubbles are excluded should be used (Atmospheric oxygen is lethal to Campylobacter fetus). Plastic pipettes can be sealed adjacent to the mucus using a pair of pliers previously heated in a flame. on dry ice is recommended. Consult with your courier if you intend to use dry ice for submission of samples.
Bacteria with particular requirements
Mycoplasma, Spirochaetes (including Leptospira, Brachyspira), Chlamydia, obligate anaerobic bacteria (including Dichelobacter nodosus) have special requirements . If culture is required, consult with your Regional Veterinary Laboratory.
Storage and despatch of specimens
In general, all samples for bacteriological examination, except smears, should be kept chilled (2-8°C) but not frozen, from the time of collection until they are received in the laboratory. This will ensure the minimum growth of contaminants. Insulated containers with frozen icebricks or plastic bags of crushed ice should be taken in the car to all investigations and specimens placed therein as soon as possible.
Delays in transport over weekends and public holidays must be considered. Specimens are better held refrigerated until the sender is sure the transport to the laboratory will not be delayed.
Samples of vaginal mucus for bacteriological examination for Campylobacter fetus should be submitted on dry ice in an insulated container. Consult with your courier.
Specimens best stored at room temperature (not refrigerated)
The following samples should be stored and transported between 18°C and 37°C (ie not refrigerated or frozen):
- Samples submitted in Campylobacter Transport Medium (CETM)
- Samples submitted in Trichomonas Transport Medium (In Pouch)
- Swabs submitted in Amies Charcoal medium for Contagious Equine Metritis (CEM)
- Swabs submitted in Amies Charcoal medium for or atrophic rhinitis.
- Tissues for fungal culture (temperatures of less than 15°C can be detrimental to fungal survival).
Despatch of specimens
See section Transport of specimens to the laboratory. In addition, when forwarding specimens for bacteriological examination the following procedures should be adopted:
- All specimens for transport should be packed in accordance with IATA requirements for diagnostic specimens (Packing Instructions 650) so as to prevent leakage and risk of accidental exposure of personnel handling the container.
- Specimens for transport from cases of suspected tuberculosis or anthrax should always be submitted individually in containers separate from all other specimens and in accordance with IATA requirements for infectious substances (Packing Instructions 602). Pack such that on opening the outer packaging, a note advising 'Suspect TB' or 'Suspect Anthrax'.
- The specimen advice form is best sealed in a plastic bag to avoid contamination or damage from water.
- Check to ensure that specimens are submitted at the temperature appropriate for the disease condition, i.e. chilled, or at room temperature. Check with your courier.