But first Your lab manual - people.upei.capeople.upei.ca/jlewis/Laboratory_schedule/WCVM-Lab... ·...
Transcript of But first Your lab manual - people.upei.capeople.upei.ca/jlewis/Laboratory_schedule/WCVM-Lab... ·...
10/01/2012
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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