Microbiology Lab Procedures

Front 1

Preparation of a Smear
(before staining)
FOR AGAR:

Back 1

Purpose:preparation of a smear from agar.
1.Wash slide several times with detergent until water does not bead off.
2.lable slide with name of the microbe.. using a "sharpe" draw a dime-sized circle in the center on the bottom side of slide.
3.Heat loop to red hot and allow to cool for 15 sec.
4.with water bottle squeeze one drop of water and "capture" drop in cooled loop.. spread the drop on circle.
5.heat loop to red hot and let cool 15 sec.
6.pass the top of test tube thru the flame to remove dust and then pass lid thru flame when taken off.
7.Slant tube and put cooled looppast the area where micro is growing and pull back very small amount.
8.flame the top of test tube the place lid on it.
9.mix material on th loop with the water on slide test with "e" test for it to be barely cloudy. Air Dry
10.with gloved hands pick up air dried slide and pass thru blue flame.. this is called "heat fixing" feel it with wrist let it burn skin with touch.

Front 2

Preparation of a smear:
(before staining)
FROM BROTH:

Back 2

Purpose: to make smear from broth.
1.lable slide with name of microbe to be stained. and draw a circle as big as a dime on bottom side of slide with sharpe.099875
2.mix liqnkasuid broth culture by gently banging againts palm15x

Front 3

Preparation of a smear:
(before staining)
FROM BROTH:

Back 3

Purpose: to make smear from broth.
1.lable slide with name of microbe to be stained. and draw a circle as big as a dime on bottom side of slide with sharpe.099875
2.mix liqnkasuid broth culture by gently banging againts palm15x

Front 4

Preparation of a smear:
(before staining)
FROM BROTH:

Back 4

Purpose: to make smear from broth.
1.lable slide with name of microbe to be stained. and draw a circle as big as a dime on bottom side of slide with sharpe.099875
2.mix liqnkasuid broth culture by gently banging againts palm15x

Front 5

Preparation of a smear:
(before staining)
FROM BROTH:

Back 5

Purpose: to make smear from broth.
1.lable slide with name of microbe to be stained. and draw a circle as big as a dime on bottom side of slide with sharpe.099875
2.mix liqnkasuid broth culture by gently banging againts palm15x

Front 6

Preparation of a Smear
(before staining)
FOR AGAR:

Back 6

Purpose:preparation of a smear from agar.
1.Wash slide several times with detergent until water does not bead off.
2.lable slide with name of the microbe.. using a "sharpe" draw a dime-sized circle in the center on the bottom side of slide.
3.Heat loop to red hot and allow to cool for 15 sec.
4.with water bottle squeeze one drop of water and "capture" drop in cooled loop.. spread the drop on circle.
5.heat loop to red hot and let cool 15 sec.
6.pass the top of test tube thru the flame to remove dust and then pass lid thru flame when taken off.
7.Slant tube and put cooled looppast the area where micro is growing and pull back very small amount.
8.flame the top of test tube the place lid on it.
9.mix material on th loop with the water on slide test with "e" test for it to be barely cloudy. Air Dry
10.with gloved hands pick up air dried slide and pass thru blue flame.. this is called "heat fixing" feel it with wrist let it burn skin with touch.

Front 7

Preparation of a smear:
(before staining)
FROM BROTH:

Back 7

Purpose:preparing a smear from broth.
1. Wash slide with detergent several times until water does not bead when it runs down.
2.lable the slide with the name of the microbe and draw a dime sized circle in the center of the slide on the bottom side of slide.
3. mix liquid broth against hand 15x use "e" test to determine how many coats you will need Very cloudy=2-5 coats or very clear broth=7-10 coats.
4. Heat loop to red hot allow to cool for 15 sec.
5. Pass the top thru the flame slanted to remove dust particles open and pass lid thru flame to kill microbes.
6. With loop obtain a loop full of broth. smear it on circle let air dry completely.Do this at least 7x and let air dry.
7.flame the test tube lid and the top.
8.after the 7th coating and the slide is completely air dry pass the slide directly thru the blue area of flame.
9.Touch slide to wrist till it burns skin.

Front 8

Preparation of a smear:
(before staining)
FROM BROTH:

Back 8

Purpose:preparing a smear from broth.
1. Wash slide with detergent several times until water does not bead when it runs down.
2.lable the slide with the name of the microbe and draw a dime sized circle in the center of the slide on the bottom side of slide.
3. mix liquid broth against hand 15x use "e" test to determine how many coats you will need Very cloudy=2-5 coats or very clear broth=7-10 coats.
4. Heat loop to red hot allow to cool for 15 sec.
5. Pass the top thru the flame slanted to remove dust particles open and pass lid thru flame to kill microbes.
6. With loop obtain a loop full of broth. smear it on circle let air dry completely.Do this at least 7x and let air dry.
7.flame the test tube lid and the top.
8.after the 7th coating and the slide is completely air dry pass the slide directly thru the blue area of flame.
9.Touch slide to wrist till it burns skin.

Front 9

The Simple Stain:

Back 9

Purpose:preparation of a smear for simple stain.
1. obtain a slide with a prepared smear.
2. lay the slide (smear side-up) on your staining rack in your sink.
3. cover the smear with Methylene Blue for 30-120 sec.
4. With water spray bottle and aim the spray above the smear and wash the excess dye off the smear Gently. Do not over wash.
5.Shake the excess water off the slide and blot it dry with bibulous paper.
6. View the slide first under 10x and then oil immersion.

Front 10

The Gram Stain:

Back 10

Purpose:This is the most important stain in micro.It is a differential stain as it seperates all microbes into two large groups.
1.place the slide with a prepared smear right side upon staining rack.
2.Cover the circled smear with the primary stain for 1 min.
3.with water bottle and gently aim the water spray above the circled smear gently washing the majority of the excess primary stain off the slide.
4. shake the excess water off the slide.
5. return the slide to the staining rack and cover the smear with the MORDANT. Immediately shake the mordant off the slide and reapply the mordant.Wait for 1 min. Shake the excess mordant off the slide.
6. Return the slide to the staining rack and drop 1-3 drops of ethanol on the smear. Quickly and immediately shack it off. Wash the smear with water as before and shake excess water off slide.
7.Return the slide to the staining rack and cover the smear with the counter or secondary stain shake it off the slide and re-apply. Leave the Counter stain on the smear for 1 min. and then wash gently.
8. Shake the excess water off the slide and blot the slide dry with bilbous paper.

Front 11

Negative Stain:

Back 11

Purpose: The simplest way to prepare bacteria for staning involves negative or background staining.
1. obtain 1 clean well washed slide and 1 dirty #2 slide.
2. draw a dime sized circle on the bottom side of the slide.
3.Add 1 drop of Nigrosine to the circled area.
4.Take a flat wooden stick and remove the plaque between the gum and tooth and rub the material on the stick onto the slide in circle mixing it with the Nigrosine.
5.Take the other slide drag the slide smearing the dye on #1.
6. Air dry and view under 10x oil immersion.

Front 12

Staining Procedure for acid Fast:

Back 12

1.Obtain a properly made smear.
2. Cover the circle with the Carbolfuchsin stain- you may use a small piece of paper over the smear.
3.Pass the slide through the bunsen burner flame until slide gets hot- about 5x.
4. Remove paper if used. wash off primary stain gently, obtain 1/2 dropper full of Acid Alcohol and spray it on circle; wash immediately.
5.Place the slide on the staining and counter stain with methylene blue.
6.wash off stain with water until the stream runs clean. blot dry.

Front 13

Endospore Stain:

Back 13

1.Obtain a properly made smear.
2. Cover the circle with the Malachite green stain- you may use a small piece of paper over the smear.
3.Pass the slide through the bunsen burner flame until slide gets hot- about 5x.
4.Remove paper if used obtain the water bottle and wash off primary stain wash until the water turns clear.
5.Place the slide on the staining rack and stain with Safranin for 3-4 min.
6.Wash off counter stain with water until stream runs clean. blot dry.