COLEGIO SAN AGUSTIN-BACOLOD

Medical Technology Program MLS 109 LABORATORY ACTIVITY SHEET

LABORATORY ACTIVITY #4
PREPARATION OF SMEARS AND SIMPLE STAINING

I. Desired learning outcomes

Staining procedures that use only one stain or dye are called Simple Staining Techniques. A simple stain
that colors the bacteria is a direct stain, and a simple stain that stains the background, but leaves the bacteria
unstained is a negative stain. Simple stains can be used to determine cell morphology, size and arrangement.

After performing this activity, students should be able to:

a. explain the importance of staining microorganisms;
b. explain the basic mechanism of staining;
c. perform proper smearing and simple direct staining techniques;
d. observe the appearance of stained bacteria under the microscope; and
e. develop patience in performing a simple direct stain.

II. Material
1. Compound light microscope 9. Specimens (any of the following):
2. Wash bottle of distilled water Gingival swab
3. Inoculating loop/needle Broth culture
4. Alcohol lamp Slant or Plate culture
5. Staining rack 10. Methylene blue stain
6. Plain slides
7. Cotton swab
8. Immersion oil

III. Procedures
SMEAR PREPARATION
1. Clean your slides well with abrasive soap or cleanser; rinse and dry.
2. Handle clean slides by the end or edge. Use a marker to make a dime-sized circle on each slide, on the
bottom of the slide so they will not wash off.
3. Label each slide according to the specimen used.
Specimen Preparation:
A. Gingival Swab
1. Using a cotton swab, collect specimen from any part of your body, preferably the mouth,
nostrils or teeth.
2. Roll the swab back and forth over contiguous areas of the glass slide to deposit a thin layer of
sample material.
B. From Broth Culture
1. Shake the culture tube and with an inoculating loop, aseptically.
2. Transfer 1-2 loopfuls of bacteria to the center of the slide.
3. Spread this out to about a ½-inch area.
C. From Slant or Plate Culture:
1. Place a loopful of distilled water in the center of the slide.
2. With the inoculating needle/loop, aseptically pick up a very small amount of culture and mix
into the drop of distilled water and spread.

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 COLEGIO SAN AGUSTIN-BACOLOD
Medical Technology Program MLS 109 LABORATORY ACTIVITY SHEET

3. Let the smears dry. Do not blow on the slides, as this will move the bacterial suspension. Do
not flame the slide, as flaming will distort the cell shapes.
4. Hold the slide with forceps and heat-fix the smears by passing the slides quickly through the
blue flame 2-3 times. Do not heat-fix until the smear is completely dry.

STAINING
1. Stain one slide at a time. Place it on a staining rack.
2. Flood the smear with Methylene blue or Crystal violet and leave for 30-60 seconds.
3. Carefully wash the excess stain off with distilled water from a wash bottle. Let the water run down the
titled slide.
4. Gently blot the smear with a paper towel or absorbent paper and let it dry.
5. Examine your stained smears microscopically using the 3 objectives. Once in the OIO, put the oil directly
on the smear. Record your observation with labeled drawings.
6. Blot the oil from the objective lenses with lens paper, and return your microscope to its proper location.
Clean your slides well.
7. Stained bacterial slides can be stored in a slide box. Remove the oil from the slide by blotting with paper
towel. Any residual oil won’t matter.

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COLEGIO SAN AGUSTIN-BACOLOD
Medical Technology Program MLS 109 LABORATORY ACTIVITY SHEET

Bacterial Smear Preparation

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 COLEGIO SAN AGUSTIN-BACOLOD
Medical Technology Program MLS 109 LABORATORY ACTIVITY SHEET

p
COURSE CODE: ________ LABORATORY EXPERIMENT NUMBER AND TITLE: _____________________________________________

NAME: ____________________________________________________________ DATE OF PERFORMANCE: ___________________

YEAR & SECTION: _____________ GROUP #: _______ TOTAL SCORE: /50

Criteria Poor Good Very Good Excellent

1 3 5 7
Readiness  Experiment set up is not  Experiment set up is generally  Experiment set up is generally  Experiment set up is prepared for
accurate, help is required workable with several details accurate with 1 or 2 small the procedure
with several major details that need refinement details that need refinement  All necessary supplies &
 Many necessary supplies  Supplies on hand but specimen  Some necessary supplies must specimen on hand
must found is lacking be searched out  Very neat and organized during
and after work
2 6 10 12
Following  Lacks the appropriate  Demonstrates general  Demonstrates good knowledge  Demonstrates very good
procedure knowledge of the lab knowledge of lab procedures of the lab procedures knowledge of the lab procedures
procedures  Requires help from professor  Carefully works to follow each  Thoroughly and carefully follows
 Often requires help from the with some steps in procedures step before moving on to the each step before moving on to
professor to even complete next step next step
basic procedures
1 4 8 10
Data  Measurements are  Measurements are somewhat  Measurements are mostly  Measurements are both accurate
Collection incomplete, inaccurate and inaccurate and very imprecise accurate with reasonable and precise
imprecise  Observations are incomplete or precision  Observations are very thorough
 Observations are incomplete recorded in a confusing way  Observations are generally and may recognize possible
or not included  Needs help of professor to complete errors in data collection
 Needs constant help of correlate and interpret results  Needs help of professor to  Able to correlate or interpret
professor to correlate or correlate and interpret results data
interprets results
1 2 5 7
Interaction No participation; sits on the  Less participation  Good participation Very good participation
with Group side with no interaction  Shows little interest  Appears interested  Is respectful of others and their
Disinterested  Doesn't pay attention to other  Enthusiastic but talks over point of view;
No stake in time group members teammates or may "hog" tasks Makes sure that everyone gets a
management  May argue to get point across  Somewhat conscious of time turn
 Helps group only when asked Conscious of time
 Fools around Gladly helps other students to
 Little emphasis on time follow procedures
1 2 4 7
Safety  Proper safety precautions are  Proper safety precautions are  Proper safety precautions are  Proper safety precautions are
consistently missed often missed generally used consistently used
 Needs to be reminded often  Needs to be reminded more  May need to be reminded once  Consistently thinks ahead to
during the lab than once during the lab during the lab ensure safety
1 2 4 7
 Proper clean-up procedures  Needs to be reminded more  Proper clean-up procedures  Consistently uses proper clean-
are seldom used than once during the lab to use generally used up procedures
Clean-up  Often requires help to proper clean-up procedures  May need some help on  Often will help other students to
complete clean-up  1 or 2 items left at station or occasion to complete tasks complete tasks properly
 3 or more items left at sink or not cleaned  Station generally left clean  Station always left neat and clean
station or sink or station not
cleaned

Instructor’s Signature: _______________________________________

Compiled by gpbdelosreyes, rmt

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 COLEGIO SAN AGUSTIN-BACOLOD
Medical Technology Program MLS 109 LABORATORY ACTIVITY SHEET

NAME OF STUDENT: ________________________________________ DATE PERFORMED: ___________________

TITLE OF LABORATORY ACTIVITY: ______________________________ DATE SUBMITTED: ____________________

IV. Observation/Results
Instruction: Paste picture/s of the bacteria you saw under the microscope. Identify their morphology
and arrangement.

Morphology ___________________________

Arrangement of cells relative to one another ____________________________

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 COLEGIO SAN AGUSTIN-BACOLOD
Medical Technology Program MLS 109 LABORATORY ACTIVITY SHEET

V. Discussion

VI. Guide Questions

1. What is the importance of simple staining?
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 COLEGIO SAN AGUSTIN-BACOLOD
Medical Technology Program MLS 109 LABORATORY ACTIVITY SHEET

2. Can dyes other than Methylene blue be used for direct staining? Why or why not?
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3. What would happen if no heat fixing were done? Or too much heat is applied?
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VII. Conclusion
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VIII. Reference/s

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