Differential Stains – Hybrid

ARRIVAL PROCEDURES

 Put on Face Mask

 Place personal belongings in cabinet near

Work Station

 Roll Up Sleeves and Tie Back Hair, if necessary

 Wash Hands

 Put on Lab Coat

o Roll up sleeves, if long

 Put on Goggles

 Put on Gloves
o Be careful not to touch your face, hair, face mask, or goggles with
gloves on
o Change gloves whenever they become contaminated

 Disinfect your Work Station

Differential Stains – Hybrid
GRAM STAIN
THIN SMEAR PREPARATION
Use frosted side of slide as TOP; Label slide
Cultures to use: Micrococcus luteus, Escherichia coli
1) Flame loop
2) Open tube, flame lip
3) Collect first sample
4) Flame lip, close tube
5) Apply sample to slide (do not spread yet)
6) Flame loop
7) Open tube, flame lip
8) Collect second sample
9) Flame lip, close tube
10) Apply sample to slide, spread into wide area
11) Air-Dry (may use slide warmer for this step)
12) Heat-Fix

Make your slide look like this:

GRAM STAIN
1) Crystal Violet (primary stain) – 1 min
2) Rinse – wash bottle, tap water
3) Gram’s Iodine (mordant) – 1 min
4) Rinse – wash bottle, tap water
5) Alcohol (decolorizer), rinse over slide just very briefly
6) Rinse IMMEDIATELY – wash bottle, tap water
7) Safranin (counterstain) – 1 min
8) Rinse – wash bottle, tap water
9) Blot dry with bibulous paper
10) Observe with microscope
11) Return microscope to 4X/Scanning lens pointing downward
12) Remove slide
Differential Stains – Hybrid
ACID-FAST STAIN
THIN SMEAR PREPARATION
Use frosted side of slide as TOP; Label Slide
1) Flame loop
2) Add loop of water to slide
3) Flame loop
4) Open tube, flame lip
5) Collect Mycobacterium smegmatis sample by generously scooping culture onto loop
6) Flame lip, close tube
7) Apply sample to slide, breaking up any clumps
8) Repeat steps 3-5 for Micrococcus luteus (one loopful), mix
9) Spread smear into wider area
10) Air-Dry
11) Heat-Fix

ACID FAST STAIN

1) Carbol fuchsin (primary stain) – 5 min, over steam, keep wet
2) Rinse – wash bottle, tap water
3) Acid-alcohol (decolorizer), briefly rinse
4) Rinse IMMEDIATELY – wash bottle, tap water
5) Methylene blue (counterstain) – 1 min
6) Rinse – wash bottle, tap water
7) Blot dry with bibulous paper
8) Observe with oil immersion
Differential Stains – Hybrid
ENDOSPORE STAIN

THIN SMEAR PREPARATION

Use frosty side of slide as TOP; Label slide
Cultures to use: Bacillus cereus, Bacillus subtilis
1) Flame loop
2) Add loop of water to slide
3) Flame loop
4) Open tube, flame lip
5) Collect sample by only gently touching growth in culture
6) Flame lip, close tube
7) Apply first sample to slide, spread in small area
8) Repeat steps 1 – 7 with second sample
9) Air-Dry
10) Heat-Fix

Make your slide look like this:

ENDOSPORE STAIN
1) Malachite green (primary stain) – 5 min, over steam, keep wet, cover with
paper towel
2) Rinse – wash bottle, tap water
3) Safranin (decolorizer and counterstain), 1 min
4) Rinse – wash bottle, tap water
5) Blot dry with bibulous paper
6) Observe with oil immersion
Differential Stains – Hybrid

CLEAN UP
 Submit Data Sheet – front lab bench

 Used Slides – Sharps Bin (red box on tabletop)

 Stains – Stain Waste Jug in Fume Hood
 Test Tubes – Remove labels and place in rack next to
autoclave
 Paper Towels – Trash

 Disinfect work area with disinfectant in spray bottle, wipe

with paper towels.
 Clean Microscope ocular and objective lenses with alcohol
and lens paper.
 Clean Microscope stage, mechanical arms, and adjustment
knobs with alcohol and paper towels
 Check that the Microscope 4x/Scanning Objective is in the
downward position

 Turn off and Unplug Hot Plate

 Lab Coat, Goggles – Plastic bag labeled with your name

 Gloves – “Gloves Only” bin beside sink

 Wash hands