Bacteriological Smear and Staining Protocol

Bacteriological Smear and Staining Protocol

Bacteria, like most cells, are essentially transparent, and must be stained in order to be easily visualized under the microscope. The specimen must be first spread out on a clean slide (preparation of a smear), heated gently to fix it to the slide, and then stained with an appropriate dye. Bacteria tend to be negatively charged, therefore positively-charged or basic dyes will bind to and stain them.

EQUIPMENT AND SUPPLIES:
clean microscope slide
soap and hot water
dH2O in dropper bottle
sample to be smeared
bacteriological loop (26 gauge Platinum as specified in Equipment for a Microbiological Work Station)
flame source
stains: 0.3% methylene blue or
Hucker’s Crystal Violet
tap water
paper towels or bibulous paper

PREPARE THE SMEAR:

01_wash_slide_P1092678sm

1. Secure a CLEAN microscope slide. The smear will not spread out properly if the slide is even slightly oily. If in doubt, wash slide well with soap and water, polish with clean paper towel or a Kimwipe (do not use brown recycled towels, they give off too much lint).

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2. a. If the specimen is solid: place a small drop of dH20 on a clean slide. Pick up small sample with a sterile loop and suspend in the water. Spread to the diameter of a dime (1.8 cm). The suspension should appear very faintly cloudy.
2. b. If liquid: place small drop of sample on microscope slide, spread to the diameter of a dime. Dilute with small drop of water if more than faintly turbid.

FIX THE SMEAR:

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3. Fix smear by lightly passing slide through a gentle flame. Do not overheat. The slide should not get too hot to comfortably hold. You are just trying to dry out the sample so that it will stick to the slide and not wash off in the next two steps.

STAIN THE FIXED SMEAR:

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4. Add a drop or two of stain (for instance, 0.3% methylene blue or Hucker’s Crystal Violet). Let sit 1 minute (or 30 seconds for Hucker’s Stain, a more powerful stain).

5. Gently rinse off excess stain with tap water for a few seconds until no more stain is seen to flow off. Gently blot dry (do not rub) with clean white paper towel. (Bibulous paper should be used if lint is a problem.)

VIEW THE STAINED SMEAR:

6. Examine with 10x objective to locate a region which is well spread and not too heavily stained. View briefly with 40x objective to confirm a well-spread, well-stained section. Then use 100x oil immersion objective to study specimen (FOLLOW Oil Immersion Protocol CAREFULLY). Adjust light for maximum clarity and carefully draw individual bacteria with all structures seen. Title the drawing as to sample, give the stain used and, at lower right, indicate the magnification of the illustrated view.

7. If you are not saving your slide, wash it well in hot soapy water, air dry in a plastic rack

Suggestions for specimens to view: Buccal smear, tooth scrapings, yogurt, buttermilk, yeast, fecal smear, vaginal smear, single colonies from agar plates, etc.

White Blood Cell Count

White Blood Cell Count

See related protocols: Blood Cell Counts, Blood Typing, Hematocrit.

White blood cells (WBC), or leukocytes, are involved in fighting infections and clearing away dead cells.  During infections, their numbers increase dramatically above the normal range of 4,800 to 10,800 WBC/cu mm.  Finding WBC content above 10,800 is suggestive of infection, or other problems (such as leukemia).

 Leukocytes are without pigment, and must be stained to be counted.  Therefore, the diluent contains crystal violet for this purpose.  It also contains acetic acid which fixes the WBC, and causes the lysis of RBC so they are not visible.  Note that a different dilution pipette is used than that in the RBC technique.

Each desk should assemble the following:

blood_letting_setup_P3130052

Image of desk set up for blood studies Equipment and supplies

Blood letting set up
2 hemacytometers
2 coverslips
one autolet
2 platforms (for drawing blood)
2 lancets (for drawing blood)
1 250 mL beaker for waste fluid
1 bottle WBC diluent (purple)
1 bottle RBC diluent (clear)
2 Kimwipes, soaked in 70% EtOH
2 paper towels

Blood cell counts can be performed using the hemacytometer. This is a precision instrument possesses a platform with microscopic grid scoring above which a specified quantity of fluid is held. By properly diluting blood, counting all cells in specified squares, and multiplying by the proper conversion factor, the number of cells per cubic millimeter can be determined.

Because of the potential dangers of working with blood, we will first practice the necessary dilutions and use the hemacytometer to count yeast cells. Be certain to master these skills before you attempt to do the blood work.

First illustrate:
1) the dilution pipets, explain their use and what the dilution factors would be
2) the grids for WBC counts
3) the grids for RBC counts.  Practice drawing water up in the pipet to the desired volume several times.
Practice drawing water up to the 0.5 and 1.0 volumes several times so that you are confident of your skill.  You will then dilute the suspensions of yeast, place an aliquot on the hemacytometer, and count all yeast in five designated squares. The convention is to count all cells touching left and bottom sides, ignore cells touching top and right sides.  When the five squares are counted, you add them up and multiply by the appropriate dilution factor (see below).

When finished for the day, wash out the pipettes and hemacytometer thoroughly with soap and water, rinse well, finish with distilled H2O rinse, replace in case.

PROTOCOL (These steps are similar to RBC, read that protocol carefully

OLYMPUS DIGITAL CAMERA

1. Swab the tip of a little-used finger with 70% EtOH.


2. Lance with quick, firm jab to the side of the pad of the finger, wipe away first blood.

OLYMPUS DIGITAL CAMERA
3. Using dilution pipet with the WHITE mixer, draw up to the 0.5 mark. Do not allow blood to congeal in pipette! Proceed immediately to the next step:

OLYMPUS DIGITAL CAMERA
4. Fill the pipet to the 11 mark with crystal violet diluent. (See below for formula.)

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5. Shake well to mix with the tip sealed with your finger.


6. Empty ~1/2 of pipet into waste container, add a small amount of the diluted blood apply to the second chamber of the hemacytometer. It should flow in to fill the chamber. (Do not over fill).
If the chamber is overfilled, quickly remove the overflow with a paper towel.


7. Let the preparation sit for a minute (for cells to settle).

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8. Examine under 100x, count the five fields indicated squares of blue-stained WBCs with a clicker (fields: top L & R, bottom L & R, center). Include in the count all cells touching left and bottom sides, ignore cells touching top and right sides.
Here are other pictures of
WBCs
WBCs
9. CLEAN UP THE EQUIPMENT: Wash out the hemocytometer, pipettes and mouth pieces thoroughly with soap and water, rinse well, finish with distilled H2O rinse, replace in case. Replace along with two pieces of hose in the case, return to the proper location in the drawer.
10. Calculate the WBCs/cmm: sum the 5 groups, multiply by 40. Enter your blood cell counts in the class data table

These are RBC views which will have to suffice until we have some WBC views for illustration…


1 Diluent for white blood cells:

10 mg crystal violet
1.0 ml glacial acetic acid
q.s. to 100 mL with d H20

Red Blood Cell Count Using a Hemacytometer

Red Blood Cell Count Using a Hemacytometer

See related protocols: Blood Cell Counts, Blood Typing, Hematocrit.

SAFETY NOTE:
Avoid infection with blood-borne pathogens such as HIV and hepatitis B, by using protective gloves when handling other peoples’ blood.  Dispose of the blood-contaminated sharps in the marked container, and the rest as indicated by the instructor.

INTRODUCTION:
Sufficient numbers of red blood cells (RBC, or erythrocytes) are necessary for adequate transport of oxygen from the lungs to the peripheral tissues.  Too few RBCs constitutes a pathological condition known as anemia (lit., without blood).  According to the Merck Manual, normal values of RBC/cmm for males is 5.4 + 0.8 million, and 4.8 + 0.6 for females.  Anemic levels for adult males are below 4.5 million, for females below 4.0 million.  We will perform red and white blood cell counts on your blood in the lab using a hemocytometer and appropriately diluted blood.

Blood cell counts can be performed using the hemacytometer. This precision instrument possesses a platform with microscopic grid scoring.  Rails on either side hold up a cover slip so that a specified quantity of fluid is held. By properly diluting blood, counting all cells in specified squares, and multiplying by the proper conversion factor, the number of cells per cubic millimeter can be determined.

PRELIMINARIES:
You should have already illustrated the following when you performed the practice counts using yeast last week:
1) the dilution pipets, with explanations of their use and what the dilution factors would be
2) the grids for WBC counts
3) the grids for RBC counts. Review the illustration of the five hemacytometer fields which you drew your notebook.

The red blood cell count may be performed at the same time as the white blood cell count and hematocrit, if you get blood to blow adequately.

EQUIPMENT SUPPLIES
2 hemacytometer kits, each with:
1 hemacytometer
1 coverslip
1 WBC diluter pipet with hose and mouthpiece
1 RBC pipet with hose and mouthpiece
1 autolet
2 lancet needless (for drawing blood)
2 platforms (for drawing blood)
microscope
squirt bottle with 70% ethanol
Kimwipes (to be soaked in EtOH)
1 bottle WBC diluent (purple)
1 bottle Ringer’s Solution (a clear diluent for RBC)
1 250 mL beaker for waste fluid
paper towel

RED BLOOD CELL COUNT:
Set up all equipment on your desk so that you are sure to have everything at your finger tips for the procedure.
Prepare an autolet with a sterile lance and platform.
1. Swab towards the side of the tip of a little-used finger with 70% EtOH. (NOT close to the cuticle!)
2. Lance by placing the platform of the autolet against the finger tip and pressing the trigger. Alternatively, us a lancet with quick, firm jab to the side of the pad of the finger. Wipe away first blood.
3. Using the dilution pipet with RED mixer from hemacytometer kit, draw blood up to the 0.5 mark. This is best done by slightly slanting the pipette down to allow blood to flow in. (Do not allow air to be drawn into the pipet or it will not draw the correct volume of blood. Slight suction should start it. (Make sure the hose is not kinked shut.) Keep the pipette level once you have filled it. Do not allow blood to congeal in pipette! Immediately proceed to the next step:
4. Continuing to hold the pipet as horizontal as possible, draw Ringer’s solution diluent up to the 101 mark. (Dilution of 1 to 200.)
5. Seal the tip with your finger and shake well to mix.
6. Empty ~1/2 of pipet into waste container
add a small amount of the diluted blood to one chamber of the hemacytometer to just fill the chamber of the hemacytometer. It should flow in to fill. (Do not over fill).
7. Let the preparation sit for a minute (for cells to settle).
8. Center the grid at 100x, switch to 400x and count and record the RBCs in each of five fields (each with 16 smallest squares) with a clicker (fields: top R & L, bottom R & L, center). Include in the count all cells touching left and bottom sides, ignore cells touching top and right sides.
Calculate the RBCs/cmm by adding the cells in the 5 groups and multiplying by 10,000 (i.e., add four zeros). Enter your RBCs/mm in the class data table.
9. Wash out the pipette thoroughly with soap and water, rinse well, finish with distilled H2O rinse, replace in case


Ringer’s Solution, per 100 mL:

860 mg NaCl
30mg KCl
35mg CaCl2

dissolve in dH2O and q.s. to 100 mL

Blood Cell Count Practice

Blood Cell Count Practice/Hemacytometer And Diluting Pipette Practice

See related protocols, Blood Cell Counts, Blood Typing, Hematocrit.

Blood Cell Count Practice

Hemacytometer with cover slip
WBC diluting pipet (white), hose and mouthpiece
RBC diluting pipet (red), hose and mouthpiece
yeast suspension:
For WBC simulation:  50 mg dry baker’s yeast + 100 mL water
For RBC simulation:   1 package baker’s yeast + 100 mL water
WBC diluent
RBC diluent
50 mL beaker for waste fluid
paper towel

Blood cell counts can be performed using the hemacytometer. This is a precision instrument possesses a platform with microscopic grid scoring above which a specified quantity of fluid is held. By properly diluting blood, counting all cells in specified squares, and multiplying by the proper conversion factor, the number of cells per cubic millimeter can be determined.

Because of the potential dangers of working with blood, we will first practice the necessary dilutions and use the hemacytometer to count yeast cells. Be certain to master these skills before you attempt to do the blood work.

First illustrate:
1) the dilution pipets, explain their use and what the dilution factors would be
2) the grids for WBC counts
3) the grids for RBC counts.  Practice drawing water up in the pipet to the desired volume several times.
Practice drawing water up to the 0.5 and 1.0 volumes several times so that you are confident of your skill.  You will then dilute the suspensions of yeast, place an aliquot on the hemacytometer, and count all yeast in five designated squares. The convention is to count all cells touching left and bottom sides, ignore cells touching top and right sides.  When the five squares are counted, you add them up and multiply by the appropriate dilution factor (see below).

When finished for the day, wash out the pipettes and hemacytometer thoroughly with soap and water, rinse well, finish with distilled H2O rinse, replace in case.

PRACTICE EXERCISE FOR LEUKOCYTE COUNT:

1. Suspend 50 mg dry yeast in 100 mL water.
2. Perform the dilution twice, add sample of each dilution to hemacytometer chambers 1 and 2 as spelled out in steps 3-6:
3. Using dilution pipet with the WHITE mixer, draw yeast suspension up to the 0.5 mark. Dab with piece of paper towel if needed to adjust volume. Proceed immediately to the next step:
4. Fill the pipet the rest of the way to the 11 mark with crystal violet diluent1. (This constitutes a 1:20 dilution)
5. Shake well to mix with the hose end sealed with your finger.
6. Empty ~1/2 of pipet into waste container, add a small amount of the diluted yeast to the first chamber of the hemacytometer. It should flow in to fill the chamber. (Do not over fill).
7.  Repeat steps 3-6 and fill the second chamber.
8. Let the preparation sit for a minute (for cells to settle).
9. Examine under 100x, count the five fields indicated squares of blue-stained yeast with a clicker (fields: top L & R, bottom L& R, center).
10. Calculate the WBCs/cmm: sum the 5 groups, multiply by 40. (Should be about 8,600 yeast cells/cmm)

RED BLOOD CELL COUNT PROCEDURE:
1. Mix a package of baking yeast with 100 mL of water, stir for 10 minutes to suspend.
2. Using the dilution pipet with RED mixer from hemacytometer kit, draw suspension yeast up to the 0.5 mark. This is best done by slightly slanting the pipette to allow the suspension to flow in. Slight suction should start it. (Make sure the hose is not kinked shut.) Keep the pipette level once you have filled it. Immediately proceed to the next step:
3. Draw Ringer’s solution diluent up to the 101 mark. (Dilution of 1 to 200.)
4. Shake well to mix with the hose end sealed with your finger.
5.  Empty ~ 1/2 of the pipet into the waste container, then add a small amount of the diluted blood to one chamber of the hemacytometer. It should flow in to fill. (Do not over fill).
6. Let the preparation sit for a minute (for cells to settle).
7. Center the grid at 100x, switch to 400x and count five fields of 16 smallest squares RBCs with a clicker (count these fields: top R& L, bottom R & L, center).
8.  Calculate the number of yeast/cubic millimeter: sum the 5 groups, multiply by 10,000 (i.e., add four zeros).
9. How many in the entire package?
10. CLEAN UP THE EQUIPMENT: Wash out the hemacytometer, pipettes and mouth pieces thoroughly with soap and water, rinse well, finish with distilled H2O rinse, replace in case. Replace along with two pieces of hose in the case, return to the proper location in the drawer.

Count as for WBC count. (this suspension is less viscous than blood)

Grids for WBC and RBC counts:

1 Diluent for white blood cells:

10 mg crystal violet
1.0 ml glacial acetic acid
q.s. to 100 mL with d H20

blood_letting_setup_P3130052

BLOOD LETTING SET UP PER DESK
13-Mar-08


2 hemocytometers, clean and polished
2 clean coverslips
2 WBC (white) diluting pipets with mouth piece and tubing
2 RBC (red) diluting pipets with mouth piece and tubing
1 bottle RBC diluent (clear)
1 bottle WBC diluent (purple)
2 hematocrit tubes, heparinized
1 crit-o-seal
2 self contained lancets
2 cotton balls
1 70% EtOH in squirt bottle
1 folded paper towel, torn in half, half per person
1 50 mL beaker for waste
1 250 mL beaker with about 50 mL dilute warm soapy water
2 clickers