clinical microbiology (dvt3224) · pdf filedesign requirements for microbiology lab ... feces)...

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).


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


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


• 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


• 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.


• 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%)


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


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)


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


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


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)


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


3. Abscess

– Collect about 3 ml of pus together with scrapingsfrom the abscess wall.


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


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.


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)


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)


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)


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



• For fluorescent antibody technique (FAT) use:

– Cell smear fixed in acetone

– Cryostat sections of fresh tissue



• 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


• Some bacteria such as salmonellae, leptospires orMycobacterium paratuberculosis may be shed intermittently

– Repeated sampling is required


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


Apparently healthy animals can be sub-clinical shedders of pathogens such as:

Salmonellae Rotaviruses Enteroviruses Coronaviruses

Leptospires………………

Feces

Urine


Diagnosis of mycotic disease apparentlycaused by Aspergillus fumigatus shouldalways be confirmed by histopathology

Invading fungal hyphae need to be demonstrated


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)

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