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Universiti Malaysia KelantanFaculty of Veterinary Medicine
Course coordinator: Dr. Erkihun AkliluLecture I (19 September 2013)
CLINICAL MICROBIOLOGY (DVT3224)
General Safety Procedures
Many of the pathogens from animals are
potentially pathogenic to human.
Careful handling
Safe disposal
Strict adherence to standard guidelines
when handling dangerous pathogens (e.g.
Bacillius anthracis).
General Safety Procedures
Design Requirements for Microbiology Lab
• Enable good management and safe laboratory
practices
• Floors and walls should be impervious to
liquids
• Work-tops should be easily cleaned
• Adequate lighting to illuminate the work space
General Safety Procedures
Design Requirements for Diagnostic Microbiology Lab
• Safety cabinets appropriate for handling of specific classes of pathogens should be installed
• Design, layout, equipment and functioning should be standard and reflective
General Safety Procedures
• Sinks should be fitted with foot-operated or elbow operated taps
• Liquid disinfectant dispensers to minimize cross-contamination
• Disposable paper towels or hot-air dryers for hand drying
General Safety Procedures
• Adequate space and facilities for sample
storage and processing, lab personnel
• Appropriate storage cabinets /rooms/space
for chemicals and samples
Safety Procedures
• Proper safety instruction for personnel workingwith potentially pathogenic microorganisms
• Protective clothing such as gowns, coats oruniform must be worn while in the lab andshould not be taken to ‘clean’ areas.
• Access to laboratories handling dangerouspathogens should be restricted.
• Eating, drinking, smoking or storage food in thelaboratory is strictly prohibited
Safety Procedures
• Mouth pipetting and licking of labels isprohibited
• All manipulations of potentially infectiousclinical material should be carried out in open-fronted, negative pressure safety cabinets or itsequivalents
• Wear disposable rubber gloves, face mask ,head cover
• Work with specimen shielded under glass orwear a visor or goggles.
Safety Procedures• Safety guidelines for storage of inflammable
materials and toxic chemicals should be strictlyenforced
• Periodic fire drills and evacuation procedures toraise awareness and enhance preparedness
• Fire blankets and fire extinguishers should bepresent in the lab all the times.
• The laboratory design should include emergencyexits which should be free of any obstruction.
• First-aid kits should be available in the labs
Safety ProceduresDecontamination
Conduct appropriate decontamination ofinfectious materials received or wastesgenerated in the lab
Contaminated materials (diagnostic specimens,media, viable cultures, glassware and surgicalinstruments) must be autoclaved at 1210 C
Use higher temperatures and longer time
Use bunsen burner fitted with a tube ordisposable plastic loops when flaming loopscaring dangerous pathogens.
Safety ProceduresDecontamination
• UV radiation can also be used (safety cabinets)
• Chemical disinfectants (in disinfectant jars)
• Frequently change disinfectant jars
• Common chemical disinfectants:– Sodium hypochlorite (corrosive)– Iodophors– Glutaraldehyde– Some phenolic compounds– Ethyl alcohol (70%)
Safety ProceduresDecontamination
Use heat-resistant autoclave bags as containers tosterlize Petri dishes and other wares
Do not put sharp objects (Pasteur pippets, needles,blades, broken glasses) directly into the plastic bags
Periodically check the efficiency of autoclaves (qualitycontrol)
Wash your lab coats and protective clothings frequently(don’t mix with other clothes)
After handling potentially dangerous pathogen autoclaveclothings before routine laundering.
Collection and Submission of Specimens
Prerequisites
• Complete case history including tentativediagnosis should be submitted to the labalong with the specimen
Collection and Submission of Specimens
General Guidelines
– Specimens should be taken from living or recentlydead animals
– Samples should be taken from the affected site(s)as early as possible following the onset of clinicalsigns
– Collect samples from clinical cases as well as in-contact animals
– Sample should be collected from edge of thelesion (including grossly healthy normal tissue)
Collection and Submission of Specimens General Guidelines
– Collect samples as aseptically as possible
– Specimen should be collected before administering anytreatment
– When possible, take generous amount of samples (blocks oftissue, biopsy material, several milliliters of pus, exudate orfeces)
– Submit specimen relevant to the problem (a wide range ofsamples can also be collected to allow flexibility and wheneverrequired)
– Submit samples individually in separate water-tight containers
– Containers must be tightly closed, labeled (Type of sample,animal ID, place and date of collection)
Collection and Submission of Specimens
Specimen for Bacteriology and Mycology (General)
1. Abortion cases
– Whole foetus (whenever possible)
– Foetal abomasal contents (ruminants), lung, liver, samples ofany gross lesion
– A piece of grossly affected placentas (and two or morecotyledons from cattle and sheep)
– Uterine discharge (if there no placenta)
– If leptospiral abortion is suspected, collect 20 ml of mid-stream urine preserved with 1.5 ml of 10% formalin
Collection and Submission of Specimens
Specimen for Bacteriology and Mycology (General)
1. Abortion cases
– Serum samples from the dam
– Placenta (cotyledons), foetal lesions, liver andlung in 10% formalin for histopathology
Collection and Submission of Specimens
2. Mastitis (Bovine mastitis)_Milk Samples
– Collect samples soon after the clinical signs arenoticed
– Do not rinse the udder with water unless verydirty.
– Thoroughly dry a wet/washed udder with a papertowel
– Wipe the udder with a cotton wool soaked in 70%ethyl alcohol (caution! with the teat sphincters)
Collection and Submission of Specimens
2. Mastitis (Bovine mastitis)_Milk Samples
– Wipe the two teats furthest from your operatingposition
– Use sterile collection tube
– Discard the first squirt of milk from each teat
– Collect milk from the nearer teats first
Collection and Submission of Specimens
3. Abscess
– Collect about 3 ml of pus together with scrapingsfrom the abscess wall.
Collection and Submission of Specimens
Specimen for anaerobic bacteria isolation
– Many anaerobes do not survive exposure tooxygen for more than 20 minutes
– Specimen from animal died more than 4 hoursprior to sampling is not suitable
– A piece of rib stripped of periosteum for bonemarrow sample ( blackleg, malignant oedema
– Samples must at the lab as soon as possible
Collection and Submission of Specimens
Specimen for anaerobic bacteria isolation
Acceptable samples:
– Blocks of tissue (~ 4 cm3 ) in sterile closedcontainer
– Tissue contained in commercial ‘anaerobicspecimen collectors’
– Liquid exudates in disposable syringes– At least 20 ml of ileal content. (when
enterotoxaemia is suspected). A loop of ileam tiedwith conents, tied of at each end.
Collection and Submission of Specimens
Urine Samples
– For bacteriological examination,• Collection by cytocentesis• By catheter• Mid-stream urine
• Clinical bacteriuria (105 bacteria/ml of urine)
• Suggestive of infection ( 104 – 105 bacteria/ml ofurine)
• Insignificant (less than 104 bacteria/ml ofurine)
Collection and Submission of Specimens
Samples for skin lesions
– Clean the surface of intact pustules or vesicleswith 70% alcohol
– Allow the surface to dry
– Aspire the contents of the lesion with sterilesyringe and fine needle
– Pluck from the lesion and scrap the edge with ablunt scalpel until blood oozes (e.g whenringworm is suspected)
Collection and Submission of Specimens
Blood cultures
Whenever bacteraemia is suspected
– Strict aseptic precaution
– Shave the area of venepuncture and clean thoroughlywith a detergent and apply 70% ethyl alcohol
– Take more than one sample within 24 hours
– Process the sample without delay (add the blood intoblood culture bottles)
Collection and Submission of Specimens
Specimens for Virus Detection
– Samples such as feces, skin scrapings, bodyfluids, tissue and blood with anticoagulants witha viral transport media
– For direct electron microscopy (highconcentration of virus): faces, lesions, biopsies,skin scrapings and vesicular fluids
– Smear vesicular fluids on glass slide, dry it andstore at room temperature
Collection and Submission of Specimens
Specimens for Virus Detection
• For fluorescent antibody technique (FAT) use:
– Cell smear fixed in acetone
– Cryostat sections of fresh tissue
Collection and Submission of Specimens
Specimens for Virus Detection
• Samples to diagnose viral respiratory diseases:
– Nasopharyngeal aspirate– Broncho-alveolar lavage– Short cotton wool sabs are unsatisfactory
• Serological tests can be used as additionalconfirmatory diagnosis.
Interpretation of Diagnostic Results
A negative diagnostic report may not be evidence of the pathogen
– Contamination (overgrown by contaminants)
– Fragile pathogens (virus /bacteria) might have died while transporting
– The animal might have stopped excreting
Interpretation of Diagnostic Results
• Some bacteria such as salmonellae, leptospires orMycobacterium paratuberculosis may be shed intermittently
– Repeated sampling is required
Interpretation of Diagnostic Results
A positive result may not confirmatory of an etiological agent’s presence
Enterobacteriaceae are ubiquitous and their detection may prove to a false positive:
Contamination by feces or soil
Postmortem invasion
Interpretation of Diagnostic Results
Apparently healthy animals can be sub-clinical shedders of pathogens such as:
Salmonellae Rotaviruses Enteroviruses Coronaviruses
Leptospires………………
Feces
Urine
Interpretation of Diagnostic Results
Diagnosis of mycotic disease apparentlycaused by Aspergillus fumigatus shouldalways be confirmed by histopathology
Invading fungal hyphae need to be demonstrated
Interpretation of Diagnostic Results
E. coli in diarroeal faecal samples of animalsmay not be significant
• Exceptions:
– Younger animals (< 10 days old)– E. coli isolates with K88, K99, F41 or 987P
fimbrial antigens (enteropathogenicity)– Pigs (susceptible to colibacillosis)