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