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But first – Your lab manual

Bring this to lab – it has:

General Lab Objectives – page II

Lab Safety: REVIEW these – page III

Basic Identification Flowcharts – p. 2-4

Basic Media & descriptions – p. 6-10

Basic Biochemical tests – p. 12-16

Gram-stain procedure – p. 17

○ Chemical Gram-stain – p. 18

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Increased awareness - why?

Increased responsibility – why?

Zoonoses, multi-drug resistant pathogens – Risk Group 2

- MRSA, MRSIG, VRE, Clostridium difficile, E. coli, NDM-1 etc.

Human Pathogens and Toxins Act (Bill C-11)

- House and Senate Approval – June 23, 2009

- In force June 1, 2009

- Amendments/updates Aug. , 2011.

- Public Health Agency of Canada

- http://www.phac-aspc.gc.ca/lab-bio/faq-eng.php

Lab 2641

Safety

- Lab Handbook – page III

- No food, no drink

- Lab coat

- No open-toed shoes

Hand Washing 101 – 7 Step

- palms, backs, interlace,

interlock, thumb/web, tips, wrists

- Wipe bench top

- Report spills

- Discard (culture/sharps)

- Avoid setting self on fire (hair back)

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Overview of Diagnostic Workflow

Next Lab

The aim of plate culture of clinical specimen is:

to ensure that bacteria present grow as isolated colonies which can be used for identification or

antimicrobial susceptibility testing.

GOOD BAD

Thanks to CAM-JG, AVC, 2011

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Copyright © Dr. R. E. Hurlbert, 1999, wsu.edu.

Thanks to CAM-JG, AVC, 2011

This is done after you have inoculated all plates.

Note: NOS = No organisms seen. You don’t always see bacteria in a direct Gram-stained smear of a specimen, even if they grow on culture. At least 1,000 bacteria/ml must be present to be detected on a smear.

Thanks to CAM-JG, AVC, 2011

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• Most media are incubated at 35-370C in air or with 5-10% CO2

• Growth checked after 18-24 hours (O/N) for fast growers – plates re-incubated for another 24 – 48 hours for fastidious bugs.

• Campylobacter spp. - microaerophilic (O2) & capnophilic (CO2 )

• Anaerobic bacteria require the absence of oxygen. (GasPak generating system or anaerobic chamber)

Thanks to CAM-JG, AVC, 2011

? ?

Thanks to CAM-JG, AVC, 2011

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1. Label the frosted end of slide with pencil.

2. With a loop, mix a small amount of a colony in a small drop of sterile water or saline. Resist the urge to make it really thick!

3. Air dry sample and gently heat fix the slide. Allow the slide to cool. Don’t overheat or you will damage the bacteria.

4. Stain with Crystal Violet for 1 minute. Rinse gently with water.

5. Apply Iodine solution for 1 minute. Rinse gently with water.

6. CAREFULLY, decolorize for 3 seconds with Gram Stain Decolorizer by flooding the slide. IMMEDIATELY, rinse with water.

7. Counterstain with Safranin for 1 minute. Rinse with water.

8. Dry slide between pages of Bibulous paper. Viol time for oil

Thanks to CAM-JG, AVC, 2011

Incomplete hemolysis (alpha)

Complete hemolysis (beta)

Double zone hemolysis (coagulase-positive Staphylococcus species, Clostridium perfringens) Thanks to CAM-JG, AVC, 2011

Blood Agar

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Pink colonies = Lactose Fermentor (LF)

Colorless colonies = Non Lactose Fermentor (NLF)

Growth = Gram-negative

Uninoculated

No Growth = *Gram-positive - Selective (crystal violet, bile salts)

*subset of Gram-negatives that do not grow – see Flowchart page 3

Enterobacteriaceae

Differential (LF/NLF) – pH indicator

Viscosity = Gram-negative

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Lab Exercises: Lab 1

Exercise 1

Loop inoculation from broth to BA and MAC.

Objective – isolated colonies

Colony morphology, hemolysis

Gram-stain each colony type – observe,

record.

Subculture - why?

Presumptive identification

antibiotic sensitivity testing next week.

Examine “under-oil”

Lab 1: Demonstration material

Bacterial Colony features

1. Streptococcus equi – Strangles in horses

2. Pseudomonas aeruginosa (NLF)

○ *MDR , opportunistic infections

3. Escherichia coli (LF)

○ Intestinal & extraintestinal (Mastitis, **UTI) infections

4. Arcanobacterium pyogenes

○ “pinpoint colonies” at 24 hours.

○ Ruminant pyogenic infections (abscess, mastitis, pyometritis)

5. Klebsiella pneumoniae (LF)

○ Mucoid, coalescing colonies, mastitis, metritis

6. Bacillus cereus

○ minor veterinary pathogen

○ Unlike it’s more serious relative B. anthracis

Microscopic Features

Fusobacterium necrophorum ○ Feedlot abscesses, footrot

Pasteurella multocida ○ Important respiratory pathogen

E. coli

Campylobacter jejuni ○ Mild enteritis in young dogs, food

contamination

Listeria monocytogenes ○ CNS infections in ruminants, food

contamination

Pseudomonas aeruginosa

*MDR = Multi-drug resistant

** UTI = urinary tract infections

LF & NLF = Lactose & NonLactose Fermentors

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Bacterial Colony terminology