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Laboratory Four: Prepping Smears and Staining For Micros

This document provides instructions for preparing smears and staining bacterial cultures for microscopy. It discusses: 1) Spreading an appropriate amount of culture on a microscope slide - too much or too little makes it difficult to see individual cells. 2) Fixing and killing the culture by heat or chemical treatment so it doesn't autolyze or wash off during staining. Drying is important before fixing. 3) Staining the smear, typically with simple stains that attach to cell walls, or diagnostic stains that reveal properties like Gram staining. 4) The provided procedure guides preparing mixed and single-culture smears from various bacteria, and staining them with Gram, acid-fast, and end

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James 'jps' Simanjuntak
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46 views

Laboratory Four: Prepping Smears and Staining For Micros

This document provides instructions for preparing smears and staining bacterial cultures for microscopy. It discusses: 1) Spreading an appropriate amount of culture on a microscope slide - too much or too little makes it difficult to see individual cells. 2) Fixing and killing the culture by heat or chemical treatment so it doesn't autolyze or wash off during staining. Drying is important before fixing. 3) Staining the smear, typically with simple stains that attach to cell walls, or diagnostic stains that reveal properties like Gram staining. 4) The provided procedure guides preparing mixed and single-culture smears from various bacteria, and staining them with Gram, acid-fast, and end

Uploaded by

James 'jps' Simanjuntak
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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Laboratory 4: Prepping Smears and Staining for Microscopy

Laboratory Four: Prepping Smears and Staining for Microscopy


When biologists study a culture under the brightfield microscope they must first prepare a smear on a
microscope slide. This involves spreading some culture on the slide, killing the culture so that it does not
autolyse and fixing it so that it does not wash off during the staining process. This process is not always as easy
as it seems.
When preparing smears is important to remember that the smallest distance that is resolvable by the
brightfield microscope is 0.2 μm. If a slide has too much culture on it then it will be difficult to see individual
cell morphology. If the smear has too few cells then it will be difficult to detect the culture under 1000X.
Spreading the proper amount of culture on the slide is critical. If you can easily see the culture on the slide
prior to staining it, then you may actually have too much culture on the slide!
After the smear is prepped it must be fixed and killed. If it is not fixed then it will wash off the slide and
if it is not killed then it may autolyse. There are two ways to do this: heat fixing or chemical fixing. Chemical
fixing involves dipping the slide briefly in methanol. Heat fixing involves passing the bottom of your slide
over a flame. Both are equally effective at killing and fixing the culture when the culture is dry on the slide. If
the culture is not dry and it is passed in the flame then the liquid may boil around the cells and cause them to
lyse. If the culture is not dry before chemically fixing then it may wash off the slide in the methanol. Drying is
important! When heating fixing it is important to not hold the slide in the flame for too long. If the slide is held
in the flame too long then the cells will burn and turn to ash. You won’t be able to see them under the
microscope.
After the smear is prepped it is usually stained so that the culture can be seen with the brightfield scope.
A simple stain is used to observe cellular morphology. Simple stains typically involve using a single staining
reagent like crystal violet, methylene blue or safranin. These stains are cationic pigments. The positive charge
is attracted to the negative charge of the cell wall. The pigments attach to the cell wall of the bacteria.
Diagnostic stains are used to stain the cells in such a way that certain properties of the cells can be determined.
All diagnostic stains involve the use of more than one reagent. Some examples of diagnostic stains are the
Gram stain, acid fast stain, endospore stain, capsule stain or flagella stain.
In this laboratory activity you will create several smears that will be stained with various procedures
(Gram Staining, Acid Fast Staining and Endospore Staining). Before reading each of the procedures below for
these activities, please read the background material provided on the three staining materials. The background
material on these diagnostic stains are provided in your GA View folder (“Background Material for Diagnostic
Stains”).

Learning Objectives for Prepping Smears


After completing this laboratory activity you will be able to
 Successfully make a smear on a slide from liquid and solid cultures that will enable you to stain and
view cellular morphology.
 Explain two different methods for fixing and killing bacteria on a slide.
 Explain why it is important to fix and kill cells before staining them.
 Describe some of the pitfalls that can occur when making smears and how to avoid those pitfalls.

Procedure
Please remember to SHARE CULTURES with the person next to you.
1. Obtain the following cultures.
 MS slant: Mycobacterium smegmatis
 SA broth: Staphylococcus aureus
 EC broth: Escherichia coli
 PA slant: Pseudomonas aeruginosa

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Laboratory 4: Prepping Smears and Staining for Microscopy

 SA slant: S. aureus
 BM broth: Bacillus Megaterium
 BS broth: Bacillus subtilis
2. Obtain the following supplies
 1 pi-pump
 2 pipettes
 1 sterile tube
 5 slides
3. Preparing your Mixed Culture: Use the sterile Pipette to aseptically transfer 3ml of SA broth to a sterile
tube. Next use a clean pipette to transfer 3 ml of EC broth to the same tube. Mix the contents of the
tube and label it “mixed” (for mixed culture). This represents what a non-pure culture looks like.
4. Prep a slide using the instructions on page 87-91 and this mixed culture. Remember to label the slide in
such a way you can figure out what you put on the slide.
5. Prep 4 more slides. To streamline things you will put two cultures on each slide (one on each end of the
slide). Look at the figures below to help you.

Follow these tips for best results:


1. Use a marker or wax pen to make a circle on the bottom of the slide in which you will apply the culture.
You cannot always see the culture on the slide until you get it under the scope so the circle really helps.
2. When preparing smears from liquid cultures
a. Mixed your cultures well. Liquid cultures tend to settle to the bottom.
b. Add 2-4 loops of culture. Use fewer loops of culture when working with extremely turbid
cultures and more loops with less turbid cultures.
3. When preparing smears from solid cultures
a. Add a drop of water to your slide first when preparing smears from solid cultures.
b. You don’t need to scrape up a lot of culture. If you can see it on your loop then you probably
have too much.
4. The pen and wax usually washes off during staining. The less you write on the slide the better. Create a
key for yourself in your notebook so you know which slide is which and label with a single number (less
to wash off and less to put back on when it does wash off).
5. Use the diagrams below to help you make your slides.

Slide 1 Slide 2
Mixed culture
Mixed EC broth BM broth

Slide 1 will be Gram Stained. Slide 2 will be Gram stained.

Slide 3 Slide 4
SA slant PA slant BS broth SA broth

Slide 3 will be Gram stained. Slide 4 will be Spore stained.


Slide 5
MS slant SA broth

Slide 5 will be Acid Fast stained.

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Laboratory 4: Prepping Smears and Staining for Microscopy

Before attempting to stain slides, be sure to read the separate handout (Background Reading for Gram
stain, Acid Fast stain and Endospore stain) about each staining procedure to understand purpose of each
staining process.

Learning Objectives for Gram Stain


After completing this laboratory activity you will be able to
 Describe how you can use KOH to predict the Gram reaction of a culture.
 Successfully perform the “string” test with KOH to predict Gram reaction of a culture.
 Successfully perform the Gram stain on a culture.
 List the reagents used in the Gram stain and state the purpose of each reagent.
 Describe why Gram positive and negative cells stain the way they do with the Gram Stain.
 Describe the cell wall structure of Gram positive and negative organisms.
 Explain what Gram variable means.
 State the color expected for Gram positive and Gram negative cells after performing the Gram stain.

Procedure
1. Read the material provided for the Gram Stain and take notes on the material as related to the learning
objectives.
2. Obtain the following
a. Slides 1,2 and 3 you prepared previously
b. A dropper bottle of KOH (share with the person next to you)
c. A new slide
d. SA slant culture
e. PA slant culture
2. Place a drop of KOH onto one end of the slide.
3. Aseptically obtain some SA culture (more than you would to make a smear) and apply it to the KOH.
Swirl it around and lift. Does a string appear? Write your observations in your lab journal.
4. Place another drop of KOH on the other end of the slide.
5. Aseptically obtain some PA culture and apply it to the KOH. Swirl it around and lift. Does a string
appear? Write your observations in your lab journal.
6. Make a hypothesis in your lab notebook about the Gram reaction of SA and PA. Which one do you
predict to be Gram positive and which do you predict to be Gram negative.
7. Obtain your slides 1, 2 and 3 that you prepared. Perform the Gram stain on each of these 3 slides. Use
the instructions in your book on pages 94-98.
8. Observe under the microscope (100X, 400X and 1000X with oil), draw and write observations in your
lab book. Write down the Stain result and the morphology of each culture.
9. Explain whether or not the Gram Stain of PA and SA met your expectations from the KOH test.
10. Look at your mixed culture. You mixed SA and EC. Now that you know what SA and EC look like
when they are Gram Stained separately, make sure you can see what a mixed culture looks like.
Distinguish pure culture from mixed culture.

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Laboratory 4: Prepping Smears and Staining for Microscopy

Learning Objectives for Endospore Stain


After completing this laboratory activity you will be able to
 Successfully perform the endospore stain on a culture.
 List the reagents used in the endospore stain and state the purpose of each reagent.
 Explain why heat must be applied when staining endospores.
 Compare the size of a vegetative cells and endospores.
 Explain why endospores and vegetative cells stain the way they do with the endospore stain.
 State the color expected for endospores and vegetative cells after performing the endospore stain.

Procedure
1. Read the material provided for the Endospore Stain and take notes on the material as related to the
learning objectives.
2. Obtain your slide #4 that you prepared. Perform the Endospore stain on it. Use the instructions in your
book on pages 112-116.
3. Observe under the microscope (100X, 400X and 1000X with oil), draw and write observations in your
lab book. Write down the Stain result and the morphology of each culture.

Learning Objectives for Acid Fast Stain


After completing this laboratory activity you will be able to
 Successfully perform the Acid Fast stain on a culture.
 List the reagents used in the Acid Fast stain and state the purpose of each reagent.
 Compare and contrast the Ziel Neelsen method of staining and the Kinyoun method of staining.
 Describe why the Gram stain cannot be used on Mycobacteria or other acid fast cells.
 Explain why carbol fuschin works well to stain acid fast bacteria.
 Explain mycolic acids and describe why the Acid Fast positive and negative cells stain the way they do
with the Acid Fast Stain.
 State the color expected for acid fast positive and negative cells after performing the Acid Fast stain.

Procedure
1. Read the material provided for the Acid Fast Stain and take notes on the material as related to the
learning objectives.
2. Obtain your slide #5 that you prepared. Perform the Acid Fast stain on it. Use the instructions in your
book on pages 99-104.
3. Observe under the microscope (100X, 400X and 1000X with oil), draw and write observations in your
lab book. Write down the Stain result and the morphology of each culture.

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