VPM 201 Veterinary Bacteriology & Mycology Fall 2014
Laboratory 2A and 2B
Introduction To
Sample Collection & Submission Isolation, Identification and Antimicrobial
Susceptibility Testing
Instructor: Dr. Anne Muckle
Welcome to Our World! AVC Diagnostic Services Bacteriology Laboratory
My teaching objective for DVM students: A basic understanding of bacterial & fungal disease
relevant to veterinary practice
AVC Class of 2012
Specimen Collection and Submission
You are the first critical step in diagnosing patient illness
Remember, it all starts with you!
• Valid culture results depend on the quality of the specimens received.
• If you have questions at the time of sampling, refer to your diagnostic lab’s lab manual, or web-page. And you can always phone the diagnostic lab for advice!
Specimen Collection and Submission
1. Collect at early infection stage and prior to antibiotic treatment.
2. Collect only from the actual infection site and use sterile technique.
3. Collect tissue or fluid preferably, otherwise use swabs with transport medium, never use dry swabs.
4. Label specimens directly and fill out a Sample Submission Form completely.
5. Store and ship specimens chilled, except for anaerobic specimens. Send to the lab within 24-36 hours properly packaged to prevent damage in transit.
Specimen Submission in Five Easy Steps
• Prepare collection site surgically to decontaminate skin prior to collecting aspirates or fluids
• Collect specimens directly from normally sterile anatomic sites (organs, body cavities) avoiding contamination by contaminants and normal flora of body surfaces (mouth, skin, conjunctiva, mucosal membranes, intestines)
• Use TTW, BALs, fine-needle aspirates, biopsy, centesis, venipuncture, catheterisation, guarded uterine culture swabs
Collect from the actual infection site Avoid sample contamination
Nasal swab or BAL?
double-guarded uterine culture swab cystocentisis
Cystocentesis sample for UTI diagnosis One organism, isolated in large numbers and in
pure culture from a normally sterile site
IDEAL Clinical Sample and Culture Result
A fine needle aspirate taken after a surgical skin prep
Hodgson JF Hughes KJ, Hodgson DR. Diagnosis of bacterial infections. Part 1: Principles of sample collection and transportation. Equine vet Educ. 2008;11:608-616.
Good Specimen Collection Techniques-Aspirates
Collecting blood culture samples:
Must do surgical skin prep of venipuncture site
Good Specimen Collection Techniques
DO NOT collect sample by swabbing the surface of wounds or inserting a swab into a draining tract. DO collect samples by fine needle aspiration or biopsy from deeper tissues (e.g. subcutaneous tissues) for open wounds, or from the actual site of infection (e.g. muscle, bone) for draining tracts.
Hodgson JF Hughes KJ, Hodgson DR. Diagnosis of bacterial infections. Part 1: Principles of sample collection and transportation. Equine vet Educ. 2008;11:608-616.
Good Specimen Collection Techniques
Good Specimen Collection Techniques - Swabs
Swab systems containing transport medium must be used so bacteria do not
dry out and die, but do not grow in transit.
• Transfer sample material into sterile containers
• Use leak-proof plastic screw-cap containers. Plastic tubes are safer than glass tubes for shipping.
• Do not use syringes and needles to transport samples (biosafety hazard!) – transfer the sample to a sterile container or transport medium swab system
• Remember biosafety of clinic and diagnostic staff – take care not to contaminate exterior of containers and swab transport systems
Good Specimen Collection Techniques - Other Specimen Containers
GOOD Sample: Plastic screw-cap
container & bagged
• Store samples for aerobic culture at 4o C prior to and during shipping.
• EXCEPT –
• ** Anaerobes samples should NOT be refrigerated; hold at room temperature.
• Blood culture bottles should not be refrigerated, ideally held at room temperature and incubated ASAP
Good Specimen Collection Techniques -Specimen Storage
• Label swabs/ containers with patient’s name AND owner’s name.
• Fill out a Sample Submission Form with patient signalment information (species, breed, sex, age); medical history and disease suspected; sample source **, any antimicrobial treatment; tests requested.
Good Specimen Collection Techniques- Labels and Submission Forms
• Ship samples ( by courier) within 24-36 hours.
• Ship specimens on ice packs (NOT bags of ice cubes!)
• EXCEPT for anaerobic culture samples and blood culture bottles NO ice packs.
• Package samples with submission forms properly to protect samples and both transport and diagnostic lab personnel. Use protective packaging - bubble wrap, bags, and solid outer containers
Transport of Specimens
• Tissues : prevent contamination, desiccation
• Blood cultures – do not refrigerate
• Joint fluid:
- will clot
- do not submit in needle and syringe, transfer to transport swab system, not a red or lavender top tube
• Dermatophytes (hairs, skin) – place in a dry container
• Anaerobic samples:
- use anaerobic or multipurpose transport systems
- submit enough sample for both aerobic and anaerobic culture set-up
- do not refrigerate
Specimens with special requirements:
BD ESwab – multipurpose transport medium tube
Specimens with special requirements:
Bovine mastitis milk samples
Source: eXtension Foundation www.eXtension.org
Good Specimen Collection Techniques
Or this!
Dry Swab – BAD! BAD because the needle is
dangerous
Don’t send this!
Send a genuine stool sample
Just a few trimmed hairs needed for dermatophyte testing
Specimen Submission Bloopers…
Maria’s Collection of Specimen Submission Bloopers…
This is what the lab will do if you send a sample like this!
Specimen Submission Bloopers
Don’t you ever do
this!
Dry swabs, sent in an envelope by regular mail
Specimen Reception at the Diagnostic Laboratory
The Diagnostic Bacteriology Laboratory
Our goal is to diagnose bacterial and fungal agents of animal disease
How?
By isolation & identification of pathogenic organisms from clinical specimens
Je suis Louis Pasteur
I am a diagnostic bacteriologist Ik ben Antoni van
Leeuwenhoek
The aim of plate culture of clinical specimens is:
to ensure that bacteria present grow as isolated colonies which can be used for identification or
antimicrobial susceptibility testing.
GOOD BAD
Specimen Culture and Identification
Species of animal
Specimen Site
History of the Animal/problem
What Bacteria?
Slide – Courtesy of Jan Giles, AVC DS Bacteriology Lab
Specimen Set-up: Submission Form
Slide – Courtesy of Jan Giles, AVC DS Bacteriology Lab
Specimen Set-up: Working in a Biological Safety Cabinet
Copyright © Dr. R. E. Hurlbert, 1999, wsu.edu.
Specimen Set-Up Swab sample
This is done after all plates have been inoculated
Gram-positive = blue/purple
Gram-negative = pink/red
Specimen Set-Up Direct Gram-stained smear
Slide – Courtesy of Jan Giles, AVC DS Bacteriology Lab
1 microlitre calibrated loop
Specimen Set-up: Urine sample
Slide – Courtesy of Jan Giles, AVC DS Bacteriology Lab
California Mastitis Test
Mastitis milk samples
Specimen Set-up: Milk sample
Slide – Courtesy of Jan Giles, AVC DS Bacteriology Lab
Selective enrichment of Salmonella from feces RVS broth
Specimen Set-up: Salmonella selective enrichment from fecal sample
Overnight RVS broth culture is inoculated onto MSRV semi-solid agar
Positive Salmonella culture: Salmonella migrate across plate
forming an opaque halo
MSRV – a semi-solid selective Salmonella medium Incubated overnight at 42o C Negative Salmonella culture:
No migration across plate
Salmonella selective enrichment followed by selective/differential culture
Post Mortem Specimens
AVC Class of 2014
Sear the surface of the tissue
Slide – Courtesy of Jan Giles, AVC DS Bacteriology Lab
Specimen Set-up Post Mortem Tissues
Cut into the tissue with a sterile scalpel
blade and insert a sterile loop or swab
Slide – Courtesy of Jan Giles, AVC DS Bacteriology Lab
Specimen Set-up Post Mortem Tissues
• Most media are incubated at 35-370C in air or with 5-10% CO2.
• Check growth after overnight incubation ( 18-24 hours) for fast growing aerobic pathogens;
• Plates are re-incubated for another 24 hours and even up to 72 hours for more fastidious bacteria.
Bacterial Culture General Incubation Conditions
• Campylobacter species require reduced O2 and increased CO2 (GasPak generating system or special incubator).
• Anaerobic bacteria require the absence of oxygen (GasPak generating system or anaerobic chamber).
Bacterial Culture Special Incubation Conditions
Mycology Mycology plates are usually incubated at room
temperature
Mycology Interesting things grow in the mycology incubator…
“You never know what’s going to greet you when you open up the incubator in the morning!”
Bacterial Culture Opening the incubator the next morning
Do you know who we are?
Bacterial Identification
Identification Procedures:
• Blood Agar (hemolytic or non-hemolytic, size, shape, odour, mucoid, swarming?)
• MAC growth (LF or NLF) or no growth?
• Oxidase, catalase reactions
• Gram stain, other stains (acid-fast, modified acid-fast)
• Set up an array of tubed biochemical tests and read reactions; compare to typical reactions of known organisms
• Refer to diagnostic text books, bacterial identification flow charts, recent publications.
Bacterial Identification
Incomplete hemolysis (alpha) Ex. Streptococcus uberis, Enterococcus species
Complete hemolysis (beta) Ex. Beta hemolytic streptococci
Double zone hemolysis (coagulase-positive Staphylococcus species, Clostridium perfringens)
Bacterial Identification Hemolysis patterns
Gram-staining is done daily in a diagnostic bacteriology lab
gram negative = pink
gram positive = purple
Bacterial Identification Staining Procedures
Hydrogen peroxide reagent
Oxidase test reagent
Bacterial Identification Two Key tests for ID flow charts
Bacterial Identification Biochemical tests
We also use commercial identification (ID) systems:
• API strip (20E, NE, Staph, Strep, Coryne)
• Remel RapID tests for aerobes and anaerobes (similar to API, but read in 4 hours)
• Biolog ID System
• ARIS Sensititre system
• MALDI-TOF Mass Spectrometry
Bacterial Identification Commercial ID Systems
API 20E
Positive Reactions
Negative Reactions
Slide – Courtesy of Jan Giles, AVC DS Bacteriology Lab
Bacterial Identification API test strips
Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
MALDI-TOF MS produces a protein spectra profile of an organism’s ribosomal
proteins for rapid identification.
Bacterial Identification MALDI-TOF Mass Spectrometry
Antimicrobial susceptibility testing must be performed as described in a
standard reference manual such as those provided by the Clinical and
Laboratory Standards Institute (CLSI)
VET01-A4: Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From Animals; Approved Standard—Fourth Edition
VET01-S2: Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From Animals; Supplement
Antimicrobial Susceptibility Testing
Disk Diffusion Method (Kirby Bauer) on Mueller Hinton Agar
Inoculum is standardised to 0.5 McFarland Turbidity Standard
= 1.5 X108 bacteria/ml
Resistant
Sensitive
Intermediate
Doxycycline
Enrofloxacin
Clindamycin
Measure the zone of inhibition (diameter in mm) compare to breakpoints for drug / organism in CLSI charts for
sensitive (S), moderately susceptible (MS) and resistant (R)
Disk Diffusion Reading the zones of inhibition
Antimicrobial Susceptibility Testing MIC Determination
MIC is the lowest concentration (highest dilution) of drug at which there is no bacterial growth
MIC Microdilution broth method Sensititre ARIS 2X
MIC Determination Antimicrobial drug test panel
Doubling dilutions of antimicrobial drugs (micrograms/ml)
MIC Determination Reading antimicrobial drug test panels
MIC Is the lowest concentration (highest dilution) of drug
at which there is no bacterial growth
MIC Report - Sensititre