Outline of Lab Session Biochemical Engineering

MAJOR ACTIVITIES OF ANY MICROBIOLOGY LAB:

o Enumeration
o Isolation
o Identification of microorganisms (generally bacteria/yeasts/moulds)

INTRODUCTION TO EQUIPMENT USED

SAFETY

o Laboratory Classification

Level Facilities Laboratory Practice Safety Equipment

1 Basic, Level 1 Good Microbiological None, work on open bench
Technique (GMT)
2 Basic, Level 2 GMT + protective clothing, Open bench + safety cabinet
biohazard signs for aerosol potential
3 Containment, Level 2 + special clothing, Safety cabinet for all activities
Level 3 controlled access
4 Maximum Level 3 + air lock entry, Class III safety cabinet, pressure
containment, shower exit, special waste gradient, double-ended
Level 4 disposal autoclave

o Routes of infection
Lungs
Mouth
Through the skin
Eyes

o Sterilization/disinfection/sanitation
Red heat (flaming)
Dry heat (hot air)
Steam under pressure (autoclaving)
Steam not under pressure (Tyndallization)
Filtration

o Rules for lab use

Laboratory wear coats/shoes/safety glasses
Never eat/drink/apply makeup
Cleanliness/decontamination
Hand washing
No personal belongings
No mouth pipetting/licking of labels
Cover all cuts
Waste disposal

ASEPSIS (for maintaining integrity of sample)

o Flaming
o Sanitizing work surface pre and post
o Working close to flame
o Sterilization of equipment

ENUMERATION OF MICROORGANISMS

o Demo of filtration

ISOLATION OF ORGANISMS

IDENTIFICATION OF ORGANISMS (example Salmonella)

o Gram stain
o Microscope demo
Gram Stain
1. Stain with crystal violet solution for 1 min.
2. Wash with tap water
3. Stain with iodine solution for 1 min.
4. Decolourise with 95% ethanol until no more stain comes away
5. Wash with tap water
6. Counterstain with safranin for 10 secs

GRAM STAIN SCHEMATIC

SOURCE: The Gram stain procedure 624 x 503 - 144k - jpg

pathmicro.med.sc.edu