Download Gram stain reagents - Bakersfield College

Microbiology Lab 4 – Staining Microbes
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Exercise 4 - Staining Microbes
Learning objectives
Following this exercise the student should be able to:
1. prepare a smear properly from broth or solid
cultures.
2. list reagents, functions and steps of a Gram stain.
3. evaluate a Gram stain reaction quality and
troubleshoot causes of Gram staining problems.
4. describe the Gram stain reaction, cell shape and arrangement of S. aureus,
E. coli and Bacillus sp.
5. interpret unknown slides for Gram stain reaction, cell shape and arrangement.
6. explain the information gathered from AFB, endospore, capsule, flagella and
inclusion body stains.
Microbes are invisible to the naked eye and difficult to see and identify even
when using a microscope. A simple stain visualizes the microorganisms; a differential
stain displays the chemical differences in cellular structures, including the cell wall
and cell membrane because the macromolecules within the structure bind to different
components of the stain. An example of this differential staining is seen in staining used
for blood smears.
Staining white blood cells with a differential stain displays the difference between
the five white blood cell types; basophils, eosinophils, neutrophils, monocytes and
lymphocytes. The intracellular granules of basophils stain dark blue because of their
affinity to basic portion of the stain Basophil means basic loving. On the other hand, the
eosinophil (acid loving) stains red as a result of the intracellular granule’s affinity to the
acidic portion of the stain. Treatment of microbial diseases depends upon the correct
identification of microorganisms and depends upon the ability to read and interpret
stained smears. Bacterial cells are commonly stained with a differential stain called the
Gram stain and protozoal cells with the Trichrome stain; this reveals the internal
structural differences and aids in identification. Properly preparing slides for staining is
important to ensure good results. Remember, you cannot see the material you are
working with so you must develop good technique based upon principles.
Always start with clean slides using lens paper to clean them. Slides can be
made from direct clinical material (a wound, sputum, knee fluid, the throat etc.), broth
cultures and from solid media cultures. The first principle is that some fluid is
needed to emulsify the material if it is dry, however too much fluid may make the
microbes hard to find. Slides from clinical cultures are usually placed directly on the
slide without the addition of water, as are slides from broth cultures. Slides from solid
media require water to emulsify and separate the individual bacterial cells for better
observation, but a very single drop of water is usually adequate. Next the material must
be attached to the slide so they don’t wash off with the staining process. The second
principle involves fixing the slide using either a chemical fixative or heat. In this
lab a heat-fixing tray will be used.
Microbiology Lab 4 – Staining Microbes
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This lab will use the principles above to make and stain bacterial slides using a
differential staining technique called the Gram stain. Initially 3 stock cultures (known
types) of bacteria will be stained, and then the 3 isolated unknown microbes from the
environmental cultures will be stained and examined. The environmental culture will
contain a variety of bacteria and possibly some fungi. Bacterial cells can be observed
for shape (rod, coccus, or spirillum) and arrangement (in chains, clusters, etc.).
Arrangements of cells are best observed from clinical and broth cultures because the
emulsification process disrupts the natural arrangement from colonies "picked" from
solid media. Read the portion in the Atlas (pages 21-34) concerning the various staining
techniques. A tutorial is also available at
http://www.microbelibrary.org/images/mayberry/gramstain/GrmStain_files/frame.htm
Materials:
Three isolation plates from lab 2
original environmental broth culture
original TSA plate
Stock cultures
S. epidermidis
E. coli
Bacillus sp.
slides
transfer loops
Gram stain reagents
Crystal violet
Iodine
Decolorizer - Acetone-Ethanol
(*note - not acid alcohol)
Safranin
Preparing the Smears
1. Collect the broth and pure subcultures you made from Ex. 2. Observe the colony
morphology. Ask the instructor to critique your isolation technique. This will be very
important in later labs. Practice the isolation technique on a new plate if you need some
more experience.
2. You will produce several smears in this lab.
 One smear will be made of each of the known stock cultures (S. epidermidis, E.
coli, or Bacillus sp.).
 One smear from your broth.
 And one from each of your pure culture plates sub cultured from the
environmental cultures (Ex. 2)
It is important to label the slides accordingly; be sure you know which side is up.
3. Put on your gloves. There are 3 steps in preparing a smear for staining. Remember to
use aseptic technique and flame the loop before and after each use.
 Preparation of the slide- Clean and dry the slide thoroughly to remove oils.
 Preparation of the smear – From the broth culture use the loop to spread one or two
drops of specimen in the center of the slide spreading it until it is approximately the
size of a nickel. When making a smear from solid media cultures, start by putting a
very small drop of water in the center of the slide and then mix a loop full of bacteria
from the surface of solid media in the water, spreading it out to the size of a nickel.
 Fixation - The point of fixation is to attach the organisms and cells to the slide without
disrupting them. In this class we will use an electric fixing tray that will dry and fix the
smears in one step. Slides must be completely dry and fixed before staining, or they
will wash off. Note - Smears made from a broth look shiny even when they are dry.
Microbiology Lab 4 – Staining Microbes
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Staining the Smears
In the beginning it is wise to make a single broth slide and a single solid medium slide and then stain and
observe these before making the other slides. This allows you to alter your technique if the results are not
optimal.
4. Begin with the known culture smears (S. epidermidis, E. coli, or Bacillus sp.). Place
the smear on the staining rack over the sink.
5. Cover the smear area with the crystal violet (gentian violet) stain and leave it for 15
seconds and then rinse the slide with a gentle stream of water.
6. Apply Gram's iodine covering the smear completely for 15 seconds and then rinse.
7. Using the Gram’s decolorizer, apply it a drop at a time to the smear area until no
more color leaves the area. (This is the most crucial and difficult step in the procedure.
Apply it evenly to the entire smear.) Quickly rinse with water to stop the decolorizing
process.
8. Apply Safranin to the smear for 15 seconds, and then rinse with water.
9. Allow the smear to air dry or place it on the drying and fixation tray.
Observing and Evaluating the Smear
10. The slide should appear only lightly colored to the naked eye. A good slide is evenly
stained and the bacteria are spread thinly enough that you can identify individual cells. The
bacteria should not be in clumps as this will alter the amount of stain retained in that area.
11. Observe the slides under oil immersion. You will be able to see little more than color
using the scanning or low power. Locate the bacteria on high power; do not use the
coarse adjustment to do this. It is of no value to try and observe bacteria on high
power. Always go to oil immersion for observation of these small organisms. *Between
slides it is not necessary to go back to low and high power. Leave the oil objective at the
same location and simply slip a new slide on the stage.
12. Observe the bacterial cells to determine their Gram reaction. These colors are hard
to differentiate at first. The purple or violet coloring is Gram positive, the pink coloring is
Gram negative. The Staphylococcus epidermidis and the Bacillus sp. should stain Gram
positive. Record your observations concerning the Gram reaction, cell shape and
arrangement on the lab report sheet. Have the instructor verify your conclusions.
Repeat the procedure for the remaining cultures.
13. Clean up.
Thoroughly clean the microscope. Discard the used slides in the sharps container at the
biohazard table. Place the cultures in the biohazard can.
14. Check out with the instructor and return your microscope to its original location.
Clean your area, wipe down the desk and wash your hands before leaving.
Microbiology Lab 4 – Staining Microbes
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Exercise 4 – Staining Microbes Lab Report
Name_________________
Lab Partners_________________
1. Have the instructor critique your isolation technique from the environmental subculture. Write down notes to improve your next isolation plate.
2. Describe the microscopic details (Gram reaction, cell shape and arrangement of the
organisms) and the macroscopic colonial details of the specimens below.
Specimen
Gram
Reaction
Cell
Shape
Cell
Colony Morphology
Arrangement Correlate the colony appearance
Gram + or
Gram -
(cocci, rods,
spirilla)
(single, chains,
clusters,
tetrads, etc.)
with the microscopic appearance
(Use terms from lab 3)
Staphylococcus
epidermidis
E. coli
Bacillus sp.
Subculture #1
Subculture #2
Subculture #3
Broth Culture
3. Using appropriately colored pencils draw the following cells.
a. Gram positive rod in chains
b. Several singular Gram negative rods
d. Gram negative coccobacilli
Microbiology Lab 4 – Staining Microbes
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e. Gram positive staphylococci, if the mordant (iodine) was not applied
f. Gram negative diplococci, if the decolorizing step was forgotten
g. Gram positive tetrads
4. During one lab period a student produced a smear that was very thick. The center of
the smear looked like Gram positive rods and the periphery appeared to be Gram
negative rods. What would you conclude about the gram reaction of these bacteria?
Circle all that apply.
a. The smear reveals the presence of both Gram positive and Gram-negative
rods in the specimen.
b. The smear reveals Gram positive rods that were uniformly over-decolorized.
c. The smear reveals Gram negative rods that were under-decolorized in the
center of the smear.
d. No conclusions can be made, the inoculum was probably too thick and the
entire procedure needs to be repeated.
5. Suppose you are viewing a clinical specimen smear made from a wound culture. You
notice that there are Gram positive cocci in clusters and Gram negative rods along with
a large number of WBC's. Circle the following assumptions that are consistent with the
smear.
a. This is the typical smear of a sterile wound.
b. The presence of WBC's indicates probable infection.
c. The fact that some bacteria stained GPC and some GNR indicates the stain
was done incorrectly.
d. The presence of GPC and GNR would indicate good staining and a wound
with a mixed infection.
e. The bacteria will need to be isolated and identified separately in order to fully
treat the infection.
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6. Fill in the chart below
Differences Associated with Gram Stain Reaction
Feature
Gram Positive
Gram Negative
Gram stain color
Cell wall
Components
Penicillin
Sensitivity
Tetracycline
Sensitivity
Toxins
Exotoxins
Endotoxins
Tolerance to Drying
High
Low
Typical examples of
Bacteria
7. Observe the clinical smears below. Draw arrows to and identify bacterial cells,
red blood cells, and white blood cells. Identify:
a.
whether they are prokaryote and eukaryote cells
b.
describe the Gram stain reaction
c.
cell shape
d.
arrangement of the bacteria.
a.
b.
c.
d.
Microbiology Lab 4 – Staining Microbes
a.
b.
c.
d.
a.
b.
c.
d.
Date last updated 8/24/2006
©Janet Fulks
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