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Name _____________________________
Period _____
Biotechnology
Biotechnology Quarter One Project
Date ___________
Manuel
This lab is designed to let students create a lab using guided instruction with minimal interference from the
teacher. Following the objectives below, students will work towards successfully using and operating lab
equipment, maintain organized and detailed observational notes, identify and correct any errors or opportunities
for improvement, and write up a proper lab report chronicling their lab experience and conclusions.
Objectives
1. Make Petrie dishes A. Hypothesize
B. Sterilize dishes
C. Mix and pour agar
D. Label and store
2. Successfully swab a surface –
A. Sterilize swab
B. Swab and bag specimen
3. Transfer swab contents to dish –
A. Handling the dish and lid
B. Proper streaking technique
C. Minimizing contamination
D. Proper Storage
4. Keep daily log of dish growth A. Monitor growth
B. Record progress
C. Make note of controllable and uncontrollable variables
D. If no growth appears, determine cause and repeat steps as necessary
5. Isolate 2 to 3 different cultures in separate dishes –
A. Make new Petrie dishes if needed
B. Sterilize loop
C. Transfer single specimen to new medium (1st pass)
D. Repeat for different strains
6. Staining and identifying isolated strains A. Identify characteristics of transferred strains
B. Use resources to identify species of organism
C. Determine if strain is common, rare, pathogenic, etc.
7. Write up lab report –
A. Follow traditional IMRAD formula
B. Be sure to address possible errors and areas for improvement.
C. Cite sources
D. Details Details Details
% Total
% Subtotal
10% of project grade
10% of project grade
10% of project grade
20% of project grade
10% of project grade
20% of project grade
20% of project grade
100% of project grade
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Quarterly Project – Lab One - Making Agar Plates
Modified from information found at http://teach.genetics.utah.edu/content/gsl/html/agar.html
Agar plates are the standard solid support material for growing microorganisms. Microbial growth media
contains nutrients and an energy source to fuel the microbes as they grow, and agar to keep the media in a semi–
solid, gel–like state.
On solid media, a single microbe will grow and divide to produce a "colony," a spot of identical descendants.
Different types of microbes produce colonies with different characteristics—shape, color, texture—which help
microbiologists determine if a culture is pure, or identify the types of microbes in a mixed sample.
A number of biological supply companies sell pre–made plates, but making your own is much less expensive.
With a little practice, you will find that it is very easy to make your own plates.
Gather supplies
 Agar power
 Distilled water
 Goggles and apron
 Masking tape and pen
 sterile, polystyrene Petri dishes 10 cm in
diameter.
 glass container that will hold at least twice
the volume of your media
 hotplate
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heat-resistant gloves, hothands, or
potholders for handling hot containers
disinfectant wipes for cleaning your work
area
graduated cylinder for measuring water
balance for weighing solid ingredients
tools for handling solid ingredients (such as
weigh boats and scoopulas, or paper plates
and spoons)
bacterial growth/isolation/identification lab report will be your own. But to prepare agar plates, you will be
working in groups of 3-4. Decide how many plates for your individual lab and then how many you will need per
group. The number of plates you will need will depend upon your hypothesis which you can develop and write
down on the following worksheet.
The instructions on the agar powder typically tell you how to make 1000 mL. This is probably way more
than you need. You will need to scale down your measurements to make the correct number of media plates.
The ideal volume for a Petri dish is 25-30 mL. We can store extra media for future use, but try not to make too
much as it would be a waste.
Prepare media
1. Use a glass container (ideally an Erlenmeyer flask) that will hold at least twice the volume of your media.
2. Measure out the amount of agar powder you will need.
3. Measure out the amount of water you will need.
4. Fill the flask with the desired amount of water and powder.
5. Heat the mixture to boiling. WARNING: The contents of the flask will come to a boil very quickly.
You must observe all safety regulations and PAY ATTENTION.
6. Make sure the agar dissolves completely. The media will take on a dark yellow translucent appearance.
7. Remove the flask from the hotplate and place on a layer or two of paper towels USING HEAT
RESISTANT GLOVES.
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8. Turn off the hotplate and unplug it. REMEMBER: THE HOTPLATE IS STILL HOT!
Pouring agar into plates
1. Prepare a suitable work area. Sterilize the table top using a Clorox wipe.
2. Place a piece of masking tape across the top of the lid so it can be labeled later.
3. Swirl the hot media vigorously to mix.
4. Let the media sit and cool for 3-5 minutes. If the media is too cool, it will start to solidify in the container. If
it is too hot, it will leave excess condensation on the lids.
5. Swirl the media again to mix just before pouring; be careful not to incorporate bubbles.
6. Pour into plate until it covers the bottom, approximately 25 mL (see video below).
7. Cover the plates with the lids.
8. After only a few minutes, the media should solidify completely.
9. Once they have solidified, tape the lid to the plate at opposite ends, keeping in mind you will need to open
and close the lids at least 1-2 more times in the coming weeks.
10. After several hours to overnight, return the plates to the plastic sleeve they came in or place them in a plastic
bag and store upside down in a refrigerator.
11. Plates will keep refrigerated for 4–6 weeks.
Tip: If you don't have a long uninterrupted chunk of time, you can prepare solid media in bottles, sterilize it, and let it cool
completely in the bottle. At a later time, you can re–heat the media in a hot water bath or microwave until it melts. Let it
cool until it is comfortable to the touch, then pour it into plates.
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Name _____________________________
Period _____
Biotechnology
Biotechnology Quarter One Project Agar Lab Worksheet
Date ___________
Manuel
This worksheet is to be handed in at the conclusion of the Agar Lab. Answer all questions, in complete
sentences where indicated.
Overarching Project Objective: Choose three areas to swab. These can be almost anywhere or anything you like;
school, home, work, clothing, personal items, etc.
Develop a hypothesis determining which of the three areas/items will be the most ‘contaminated’, that is, which
will bring about the most growth.
Write your hypothesis below in a complete sentence or sentences.
1. Hypothesis:
During the lab you will most likely need to scale the amount of agar you make. Use the space below to show
your conversion work.
2. # of Petri dishes required for your individual lab _____
3. Agar and H2O amounts required by the instructions: Agar _________ Water _________
4. Converted rates based on your groups needs: Agar _________ Water _________
5. Did you successfully complete the lab by following all instructions and safety guidelines? _________
6. Did you make enough agar to fill your requirement? _________
7. Did you have any excess agar? _________ Approx. how much? __________
8. What errors or areas of improvement did you recognize during the lab? Write your answer in complete
sentences.
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Name _____________________________
Biotechnology
Period _____
Date ___________
Manuel
Quarterly Project – Lab Two – Swabbing, Plating, and Observing
Use a sterilized inoculating loop or sterile swabs to collect bacteria from different locations and then streak each
petri dish with your sample. This involves a bit more technique than Experiment 1 but offers a wider choice of
bacteria sampling locations. Swabs can be run over doorknobs, bathroom fixtures, animal mouths, etc.
What you need.
 Prepared petri dishes containing agar medium and nutrients.
 Bacteria collected from doorknobs, bathroom fixtures, etc.
 Pen or pencil for labeling bags and dishes.
 Masking tape.
 Sterile swabs
 Sterile gloves
 Disinfecting wipes
 Observation notebook
Collecting your Samples
1. Once you have decided which three areas to swab, collect the following materials:
a. Sterile gloves
b. Sterile swabs
c. Sterile baggies
2. Label each bag into which you will place your collected sample.
3. Keep the swabs closed until you are ready to collect your sample.
4. Wearing the gloves, remove the swab.
5. Rolling the swab between your thumb and forefinger, run it over the surface of the area you wish to test.
a. If your swab is double sided, use both ends to ensure your plate your sample on the Petri
dish.
6. Immediately bag and seal your swab to avoid contaminating the sample.
You may use the space below to record your swabbing information and in the field to record any problems or
unexpected issues.
Field Notes:
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Plating and Observing your Samples
Prepared petri dishes should be refrigerated until used and always stored upside down (i.e media in upper dish,
cover on bottom). This keeps condensation which forms in the lid from dropping onto and disrupting the
bacteria growing surface.
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1. Once you are ready to plate your samples collect the following materials:
a. Sterile gloves
b. Sample swabs
c. Sanitizing wipes
d. Prepared Petri dishes containing agar medium and nutrients.
e. Pen or pencil for labeling bags and dishes.
f. Masking tape.
When ready to use, let dishes come to room temperature before taking samples (about one hour).
Unseal your dish.
Remove the lid only as far as you need to apply your sample. Try not to remove the lid completely. You may
need a partner to help you with this.
Inoculate each dish by streaking a pattern gently across the entire agar surface without tearing into it.
Another common technique is to divide each plate into four quadrants by marking the lid with a cross.
A. Streak your sample in straight lines starting in quadrant 1.
B. Generally, after a few days, quadrant one will show the most growth.
C. Depending on bacteria abundance on the swab, quadrant 4 may show no grow or only a few
colonies.
D. It is sometimes easier to distinguish different bacteria types in this low growth, less cluttered area.
Replace cover on dish, tape closed, and label each dish so you know the source of the bacteria. Store upside
down.
Dispose of your swabs properly.
Let grow in undisturbed warm location, ideally in an environment around 100° F (37° C) - not in sunlight or
on a heating register.
You should see growth within a couple of days. The dishes will start to smell which means the bacteria are
growing.
Make observations and keep records of what you see growing in each dish. Record any conclusions or
unexpected obervations
Before disposing of dishes in the trash the bacteria should be destroyed. Pour a small amount of household
bleach over the colonies while holding dish over sink. Caution - do not allow bleach to touch your skin, eyes
or clothes. It will burn!
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SUBCULTURING MICRO-ORGANISMS
Source: NQ Curriculum Support Intermediate 2 Biotechnology
(Unit 2 Student Materials)
In the laboratory, micro-organisms are usually grown or cultured in liquid medium (broth) or on solid medium
(agar plates or slopes). Growth of bacteria and yeasts shows as cloudiness or turbidity in the broth although
sometimes bacteria grow as a layer on the surface of the broth or at the bottom of the culture tube.
The growth on plates depends upon how the plate has been inoculated.
Types of inoculations on agar plates
Subculturing is the aseptic transfer of micro-organisms from a culture to fresh medium. The freshly inoculated
medium is then incubated at the temperature appropriate for growing the organism.
There are four subculturing procedures with which you should become familiar. They are:
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Solid to solid: the transfer of bacteria or fungi from an agar slope or plate culture to an agar plate
Solid to liquid: the transfer of bacteria or fungi from an agar slope or plate culture to a broth
Liquid to solid: the transfer of bacteria or fungi from a broth culture to an agar slope or plate
Liquid to liquid: the transfer of bacteria or fungi from a broth culture to a broth
Containers of culture media to be inoculated must be labelled with initials, date and name of organism. To
prevent possible confusion, plates are marked on the underside while tubes and bottles must be labelled on the
side. Lids are not labelled.
The use of aseptic technique minimises the risk of contamination of cultures and also reduces the risk of
micro-organisms from the laboratory cultures escaping to the environment.
You will use a wire loop, a straight wire, forceps or a scalpel to subculture bacteria or fungi or to transfer
specimens to a slide. The wire loop is the basic tool of the microbiologist and you should learn to handle it
correctly.
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Flame Sterilisation of Instruments
Metal instruments used to transfer micro-organisms are sterilised by heating till red hot in the blue part of the
Bunsen flame heat. They must be heated till red hot to make sure that any contaminating bacterial spores are
destroyed & this process must be carried out before and after use.
Good flaming technique is very important to
avoid contamination of the surrounding air with
aerosols.
A Bunsen burner is lit and the air hole opened fully
to provide a blue flame. The operator holds the
loop between thumb and fingers as if holding a
pencil very loosely, at an angle that is almost vertical.
The loop is placed in the light blue cone of the
flame. Positioning the loop in this cool area of the
flame allows any liquid to dry out and prevents
formation of aerosols. Aerosols are fine liquid or
solid particles that are dispersed into the air and
might contain micro-organisms.
Placing a loop in blue cone of flame
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After any liquid material has evaporated, the loop is
drawn slowly up into the hottest region of the flame
(immediately above the light blue cone) and held
there until it is red hot.
Drawing loop into hottest region of flame
The loop is then withdrawn from the flame and
allowed to cool before touching micro-organisms.
Cooling the loop
Straight wires may be sterilized in the same way.
Aseptic Transfer Operations
Once the loop has been sterilised and cooled, it is used to remove organisms (the inoculum) from a culture and
inoculate sterile growth medium. When working with micro-organisms, aseptic techniques are used to avoid
escape of organisms to the environment.
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To minimise the chances of contamination, cultures and media are exposed to the air for the minimum time it
takes to perform a manipulation or to make observations. All subculturing procedures are carried out close to a
Bunsen flame.
After flaming, the loop is not put down until the procedure has been completed.
Lids of Petri dishes are never completely removed. They are opened just enough to allow entry of the loop
to perform the manipulation and minimise exposure to the air.
Liquid cultures are disturbed as little as possible to reduce the risk of aerosol formation. Lids from cultures
are never placed on the bench surface where contamination might occur. They are removed from the bottle
or tube using the little finger, held there while manipulation of the culture takes place and then replaced. The
rest of the hand is free to carry out the manipulation. Lids are tightened to prevent spillage before incubation.
To remove any contaminating organisms on the neck of a bottle, it is passed through a hot Bunsen flame on
removal and before replacement of the lid.
The loop must be flamed to red heat when the subculturing procedure is finished.
The table below summarises potential points of contamination and the techniques employed to minimise the
risk.
Contamination Risk
Precaution
Inoculating loop
Flame and cool.
Do not lay down loop until procedure is
complete.
Work close to Bunsen flame.
Opening of Petri dish (solid medium)
Open lid for as short a time as possible.
Open lid just enough to insert wire loop.
Opening of liquid cultures (bottles, tubes)
Hold the lid in crook of little finger – never
place on work bench.
Pass neck through a hot Bunsen flame
before insertion and after withdrawal of
loop to kill any contaminating organisms.
Incubation of cultures
After inoculation, cultures are incubated at a given temperature. Petri dishes should be placed upside down to
prevent condensation dropping on to cultures. The lids of Petri dishes should be secured to the base with
diagonal strips of sellotape. Bottles should be stored upright in a container which will prevent them being
knocked over.
Subculture results
Subculturing has been successful when the transferred organisms have grown in the new medium without
contamination.
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Contaminating organisms can usually be observed on plate cultures as single colonies with a different
appearance. In a broth culture, it is impossible to tell by simply observing the broth whether the culture is pure.
The liquid culture must be streaked out on an agar plate to determine if there are any contaminants.
In the practical work associated with this course, you will generally be working with known organisms and with
pure cultures. A pure culture is one in which there is only one type of organism.
STREAK PLATES
Source: NQ Curriculum Support Intermediate 2 Biotechnology
(Unit 2 Student Materials)
When ‘plating out’ micro-organisms various techniques can be used. However, when attempting to obtain
isolated colonies the streak-plate method is routinely used.
Streak Plate Inoculation: Solid to Solid
Instructions
These instructions are for right-handed people. If you are left handed, please reverse the instructions
accordingly.
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Wear a lab coat.
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Prepare your work space on the bench, collect the materials and set them out correctly on the bench.
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Label the bases of the Petri dishes containing sterile nutrient agar with initials, date, name of micro-organism
and S
S (for solid-to-solid transfer).
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Hold the loop in the right hand.
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Flame the loop and allow to cool.
Do not put down loop or wave it around.
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Lift the lid a little of the Petri dish containing the inoculum with the left hand.
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Touch a single colony with the wire loop.
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Withdraw loop. Do not put down loop or wave it around!
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Replace lid of Petri dish.
10 Partially lift the lid of the Petri dish containing the solid medium.
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Holding the loop parallel with the surface of the
agar, smear the inoculum backwards and forwards
across a small area of the medium. Replace lid.
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Flame loop and allow to cool.
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Turn plate. Streak loop from A across the surface of
the agar in three parallel lines. Replace lid.
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Flame the loop and allow to cool.
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Turn plate. Streak loop from B across the surface of
the agar in three parallel lines. Replace lid.
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Flame loop and allow to cool
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Turn plate. Streak loop from C to D across the
surface of the agar as shown, without touching area
A.
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Replace lid of Petri dish
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Flame loop
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Incubate plate upside down at 30C for 72 hours
SMEAR PLATE: SOLID TO SOLID
Source: HSDU Biology and Biotechnology Microbiological Techniques Intermediate 1- Advanced Higher Folder
1
Hold the loop in the right hand.
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Flame the loop and allow to cool. Do not put down loop.
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With the left hand, lift the lid a little of the Petri dish containing the inoculum.
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Touch a single colony with the wire loop.
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Withdraw loop carefully without touching plate. Do not put down loop or wave it around.
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Replace lid of Petri dish.
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Partially lift the lid of the agar plate and gently rub the loop across the surface of the agar (see diagram
below). Take care not to break the agar surface.
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Replace lid of Petri dish.
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Sellotape lid to base of plate (two diametrically opposed pieces).
10 Invert and incubate under appropriate conditions.
SPREAD PLATE
Source: HSDU Biology and Biotechnology Microbiological Techniques Intermediate 1- Advanced Higher Folder
Spread plates, also known as lawn plates, should result in a culture spread evenly over the surface of the growth
medium. This means that they can be used to test the sensitivity of bacteria to many antimicrobial substances,
for example mouthwashes, disinfectants and antibiotics.
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Loosen the lid of the bottle containing the broth culture.
Hold a sterile pipette in the right hand and the bottle/test tube containing the broth culture in the left.
Remove the lid/plug of the bottle/test tube with the little finger of the right hand and flame the neck.
With the pipette, remove a small amount of broth.
Flame the neck of the bottle/test tube and replace the lid/plug.
With the left hand, partially lift the lid of the Petri dish containing the solid nutrient medium.
Place a few drops of culture onto the surface [about 0.1 cm3].
Replace the lid of the Petri dish.
Place the pipette in a discard jar.
Dip a glass spreader into alcohol, flame and allow the alcohol to burn off.
Lift the lid of the Petri dish to allow entry of spreader.
Place the spreader on the surface of the inoculated agar and, rotating the dish with the left hand move the
spreader in a top-to-bottom or a side-to-side motion to spread the inoculum over the surface of the agar.
Make sure the entire agar surface is covered.
This operation must be carried out quickly to minimize the risk of contamination.
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Replace the lid of the Petri dish.
Flame spreader using alcohol.
Let the inoculum dry.
Seal and incubate the plate in the inverted position.
SUBCULTURING MOULDS USING i) SCALPEL ii) CORK BORER OR BLUNT END OF
PASTEUR PIPETTE
Source: HSDU Biology and Biotechnology Microbiological Techniques Intermediate 1- Advanced Higher Folder
1
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3
4
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Dip the implement in ethanol, place in Bunsen flame briefly and allow ethanol to burn off. Do not put
down.
Keep the ethanol at a safe distance from the Bunsen flame.
Lift the lid a little of the Petri dish containing the inoculum with the left hand.
Hold the inoculating implement in the right hand.
i) with scalpel, cut a square shape in the fungal mycelium;
OR
ii)
with forceps, pick up a little piece of mycelium;
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OR
iii)
with cork borer or pipette, cut a core of mycelium;
(if the centre of the colony is producing spores, remove the inoculum from the edge of the colony).
Remove a cube, strands or core of agar with its fungal mycelium
Withdraw implement from Petri dish.
Replace lid of Petri dish.
At all times, hold the inoculating implement as still as possible.
Partially lift the lid of the Petri dish containing the sterile malt agar medium.
Place the cube/bore of agar or fungal mycelium on to the centre of the sterile agar. Use a mounted needle
which has been flamed in ethanol to assist with the transfer process if necessary.
Withdraw implement.
Dip the implement in ethanol, flame as above and place on heat resistant mat.
Staining and Interpretation of Smears
[Preparing a smear] [Gram stain procedure and examination] [Negative staining] [Spore staining [Observation of
living bacteria]
Important information such as shape and degree of motility can be obtained by observation of living bacteria
with the phase contrast or dark field microscope. However bacteria are routinely stained with different dyes in
order to reveal different properties and to enhance contrast for viewing with conventional bright field
microscopy. A number of stains have been developed to distinguish spores, nuclear bodies, capsules, and
characteristics of the cell wall.
The staining methods we will use kill the bacteria, reducing the risk of infection by pathogenic organisms. Since
the rigid cell walls of bacteria prevent distortion of morphology upon drying, samples can be spread onto a glass
slide and air dried, then fixed to the surface by passing the slide quickly through a flame, melting the complex
carbohydrates of the cell walls to the glass and killing the cells.
The Gram stain is routinely used as an initial procedure in the identification of an unknown bacterial species.
Bacteria bear a slight net negative charge and usually bind positively charged dyes such as methylene blue and
crystal violet. A species can be classified as Gram positive, Gram negative, or Gram variable depending on the
ability if cells to retain the blue dye. Gram negative bacteria do not retain the dark blue color, but can be
counterstained a light red so that they can be seen in bright field microscopy. Since two dyes are used to
distinguish types of bacteria, Gram staining is called a differential staining method.
The Gram stain is a direct method, since the cells themselves retain dye. In indirect, or negative, staining, smears
are produced by mixing material with India ink or acidic dyes such as nigrosine. Acidic dyes have a negative
charge and are repelled by the negatively charged cell walls. Cells remain unstained against a dark background.
Some species produce spores, which are dormant cells with thickened cell walls. Spores are often detectable in
Gram stains or by phase contrast microscopy of living cells, however differential staining methods may be
necessary to confirm or reject the presence of spores in a culture. As with the Gram stain, a spore stain
distinguishes spores on the basis of cell wall properties.
In the laboratory you will practice the Gram stain technique on a variety of Gram positive and Gram negative
bacteria grown on different types of media. You will learn the technique of negative staining with nigrosine dye
in order to clearly distinguish shapes of bacteria. You will also carry out spore staining on appropriate cultures.
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Finally, you will observe living bacteria to become familiar with features that can be seen without staining,
including cell shapes, patterns of association, and motility.
Preparing a smear
A properly prepared smear accomplishes two things. It causes bacteria to adhere to a slide so that they can be
stained and observed. It also kills them, rendering pathogenic bacteria safe to handle. An objective in preparing
smears is to learn to recognize the correct density of bacteria to place on the slide. Too many, and they overlap
each other giving false positives or crowding each other to make a mess. Too few, and they cannot be located on
the slide.
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A circle should be marked on the under side of a slide with a glass etching tool. Several circles can be
located on the same slide.
The slide must be grease-free. A good way to clean a slide is to repeatedly breathe on it, followed by
rubbing vigorously with a Kimwipe or paper towel to remove the fog. When the slide de-fogs
immediately after breathing on it, it is sufficiently clean.
To prepare a smear from a dry culture, a very small drop of distilled water should be placed over the
circled area. After aseptically removing material from a culture it is them mixed with the drop or placed
directly on the slide if it is a dilute broth culture. It takes very little material to produce a successful
smear.
The drop is air-dried completely, which takes a short time if a small drop is prepared.
While holding the slide with a clothes pin it is quickly passed it through a flame. Three quick passes are
usually sufficient to kill the bacteria and cause them to adhere.
After cooling the slide, the staining procedure is conducted.
How to screw up a smear
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Forget to clean the slide
Use too much material - suspension should be just barely cloudy
Use so much liquid that it takes forever to dry
Heat the smear before letting it air dry, boiling the bacteria instead of attaching them
Overheat the smear, melting cell walls and possibly breaking the slide
top
The Gram stain
In Gram staining bacteria fixed to a slide are treated with a basic dye that binds electrostatically to the negatively
charged cells. Next, the preparation is treated with a mordant such as iodine to form an insoluble dye-iodine
complex. The slide is then washed with alcohol to solubilize and remove the dye-mordant from Gram negative
cells but not Gram positive ones. Differential extraction of the dye-mordant by the decolorizing agent is the
critical step that distinguishes the bacteria. A counterstain, safranin, is applied in the final step. Cells that have
been decolorized will take up the second basic dye whereas those already stained with the first dye will not.
The mechanism of the differential staining response has not been resolved with certainty. One theory holds that
differences in the cell wall chemical composition account for the staining response. A second theory maintains
that the thicker walls of Gram positive bacteria are dehydrated by the decolorizing solution and shrink, resulting
in the closure of pores in the wall, trapping the dye-mordant within the cell. The thinner cell wall of Gram
negative bacteria would be readily penetrated by the decolorizer. What is known with certainty is the critical role
16
of the cell wall. Removal or alterations of the wall from Gram positive organisms converts them to Gram
negative cells.
Since many Gram positive bacteria tend to become Gram negative with age, the Gram stain should be used with
overnight cultures. Sample from the edge of a colony, where cells are actively growing.
The stains you use in this laboratory will stain hands and clothing as well as bacteria. Do not wear good clothes
in the laboratory, wear gloves when staining, and keep in mind that all stains are assumed to be toxic unless
otherwise stated.
Components
Different formulas have been used for crystal violet, safranin, and decolorizer, all of which are effective.
1. Gram's crystal violet: 1% aqueous crystal violet dye; [Hucker's crystal violet] 2 g crystal violet 90% dye
content, 20 ml ethyl alcohol, 0.8 g ammonium oxalate, 80 ml distilled water
2. Gram's iodine: 1 g iodine, 2 g potassium iodide, 300 ml distilled water
3. Gram's safranin: 4 g safranin powder, 200 ml ahydrous ethanol, 800 ml distilled water
4. Gram's decolorizer: 25% acetone, 75% isopropyl alcohol
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A heat-fixed bacterial smear is first covered completely with a few drops of a solution of crystal violet, a
purple basic dye.
After 30-60 sec the smear is rinsed with water by squirting the slide above the smear and letting the water
wash over it until the water runs clear.
Several drops of iodine (the mordant) are applied to cover the smear and left for 60 sec., then rinsed
again.
A few drops of an isopropanol-acetone mixture or similar solvent are added at a time until the wash is
colorless, then the slide is rinsed again.
A red basic dye, aqueous safranin, is applied for 30-60 sec.followed by a rinse.
The smear is blotted (not wiped) to remove excess water, using bibulous (absorbent) paper or a paper
towel. The slide is then air dried the rest of the way.
How to screw up a Gram stain
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Screw up the smear to start with
Apply stain to the wrong side of the slide
Stain or rinse the smear incompletely
Allow stain to dry up before rinsing
Forget to decolorize or decolorize incompletely
Rub off the material when you blot it
Microscopic observation of a Gram stain
A good approach to observing any smear of very minute objects is to examine it under low power (40x) in bright
field to become oriented. After focusing, one then works up to 100x, 400x, and finally oil immersion (100x). The
immersion oil is placed directly on the smear. By the way, the focal lengths of the high dry and oil immersion
lenses are less than the thickness of a slide. The smear must be up, or focusing won't be possible. The top of the
slide can be identified by feeling for the etched circle on the bottom of the slide.
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At low magnifications Gram stained material looks like dirt on the slide. Higher magnifications are needed in
order to see any detail at all. Bacteria are often concentrated in a ring around the original smear. Bright field oil
immersion microscopy is necessary to see an undistorted image of any directly stained bacterial smear. A Gram
negative or positive phenotype cannot be confirmed with certainty using only a dry magnification, since typical
cells are a half micrometer in diameter, less than the best resolution of the high dry lens. Phase contrast or dark
field viewing improve the resolution, but both distort the color. High dry magnification distorts both shape and
color.
After putting immersion oil on a slide, the high dry (40x) lens can't be used again unless the oil is removed. Slides
are usually blotted to remove excess oil, then dipped in xylene several times to dissolve the oil film, and air dried
in the fume hood.
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Indirect (negative) staining
The acidic dye nigrosine will be used to visualize the capsule or sheath that surrounds some bacteria in a process
called negative staining. Capsules are composed primarily of polysaccharides or glycoproteins and are gelatinous
in texture. They are readily destroyed by heating and hence direct staining methods cannot be utilized. In general,
the size and shape of microorganisms is often less distorted with indirect staining procedures, especially when
sampled from a broth culture. Therefore negative staining is useful whenever the morphology of individual
bacteria is in question. Morphology can often be determined with confidence with only the high dry lens.
Consider that this procedure does not necessarily kill the organism, so be careful.
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After preparing a clean, greaseless slide, a small drop of nigrosine is mixed with a small drop from a
broth culture or with a quantity of dry material.
The drop is spread across the slide using the edge of another slide as a spreader. This same procedure is
used for blood smears.
After air drying, the smear is observed using the high dry lens, or oil immersion if desired. The smear will be
most dense where the nigrosine dye was deposited on the slide. Somewhere along the tapered spear the density
will be ideal. The background should be blue-gray. Bacteria will be evident by the absence of any color.
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Spore staining
The following modification of the Wirtz method, described below, has been effective and trouble free in our
experience. You are cautioned, though, that the color may fade after a few days. The result is a delicate
combination of green and red that is readily recognized provided lighting is optimum. ***CAUTION***
Malachite green dye is toxic, and breathing of fumes or contact with skin can be hazardous. This procedure
should be carried out in a fume hood. The stand, burner, dyes and pipets will be available in the hoods. The
procedure is messy - wear a lab coat or old clothes.
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A smear is prepared and fixed with 20 passes through a flame.
The smear is placed on a screen well above a moderate flame from a bunsen burner.
A generous amount of saturated aqueous malachite green is applied to the slide and allowed to steam off
for 10 to 20 minutes. Ideally, the heat is maintained so that the dye barely steams, that is, whitish vapors
are barely visible. Dye must be added so that the liquid does not dry up.
After cooling the slide is rinsed with tap water to remove excess stain.
The slide is then counterstained for a minute in 0.25% aqueous safranin. The slide is then rinsed, blotted,
and dried.
Spores stain a light green,while the rest of the cell stains pink. Spores are best seen with oil immersion
microscopy. Often, the colors are not very strong, so it is necessary to have the microscope in good alignment
with optimum contrast and lighting. Make color notes right away, as the green may fade after a few days.
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Observations on living bacteria
Sometimes assay results are compromised because a contaminating organism grows in the medium instead of the
intended bacterial isolate. For a quick check to verify that cell morphology is consistent with the culture from
which the inoculum was taken, a wet mount can be prepared and examined in dark field and/or phase contrast.
If present, endospores are often evident in phase contrast, allowing one to avoid having to do a spore stain.
Very often, identification of an unknown organism requires knowledge of its motility, that is, its capability for
translational movement. The results of motility agar incubations can be difficult to interpret, partically for
aerobic bacteria that don't grow well deep into the agar. A good quick check for motility is to examine a very
young culture using the hanging drop method. A young culture would be a broth culture inoculated the night
before, or a broth culture that was diluted 10 fold or so in the morning, incubated, and examined in the
afternoon. A hanging drop culture is prepared by placing a very small drop of medium on a coverslip, then
inverting the coverslip over a depression slide so that the bottom of the drop does not make contact with the
slide itself. Vaseline can be used if necessary, to make a sealed chamber.
Hanging drops can be examined using all objective lenses, although to be able to look throughout the depth of
the drop the limit may be 100x. The curved depression slide will distort the effects of phase contrast, but dark
field may work and will be sufficient to detect movement. All live bacteria move by Brownian (molecular)
motion, at a vibration rate that is inversely proportional to the size of the cell. Rapid Brownian movement is a
common characteristic of non-motile cocci such as Staphylococcus, Streptococcus, or Micrococcus. However
some bacteria are flagellated, and exhibit translational movement as well. Truly motile organisms will zip across
the microscope field. Look for definite directional motion, tumbling, and movement against currents.
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Dispose of wet mounts carefully, since the bacteria will be viable.
Staining of Fungus by using Lactophenol cotton blue
Staining of Fungus by using Lactophenol cotton blue
Aim
To stain fungal cells by using Lactophenol cotton blue.
Approach
Fungus are eukaryotic organism and they are classified into two main groups that is yeast and molds.Its cell wall
is made up of chitin.Fungal cells have both macroscopic as well as microscopic structure .This micro-organism is
useful as well as harmful to human being useful because it produces many antibiotics,natural products as well as
used in industrial fermentation process.It is harmful in the sense as it causes human diseases,produce toxic
substances as well as harm important crops. So it is very important to study fungal species.The branch of science
that deals with study of fungal species is called as Mycology.Staining of these fungal cells is a very important
step.These are some examples of fungi Rhizopus, Aspergillus, Penicillium, Fusarium, Candida,Mucor etc.Here we are
going to stain fungal cells by using Lactophenol cotton blue method this staining technique is also called as
mounting of fungus.
Requirement
1.
Young fungal culture
2.
Glass slide
3.
Cover slip
4.
Needle
5.
Lactophenol cotton blue.
Procedure
1.
Take a clean grease free slide.
2.
Add a drop of mounting fluid that is lactophenol cotton blue solution on a slide.
3.
Sterilize the needle and cool it then transfer a mycellial mat on fluid and press it gently so that it easily
mix with the stain.
4.
Take a clean cover slip and with the help of a forcep place the cover slip on mycellial mat.
5.
Take a blotting paper and wipe the excess stain .
6.
Observe under low to high power objectives of microscope.
Mechanism
Here in this mounting method we use Lactophenol cotton blue stain where Lactophenol acts as a cleaning agent
as phenol is fungicidal in nature so it kills the organism.Lactic acid acts as a preservative and it preserves the
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structure of fungus.where as cotton blue stains the cytoplasm in light blue colour so here we can observe the
fungal structure colourless with light blue background.
Observation
After staining fungus cell we can observe different parts of a cell like conidia, hypae as well as conidiophore etc.
Result
On basis of observation the type fungal organisms identified.
Interpreting plates
http://www.sciencebuddies.org/science-fair-projects/project_ideas/MicroBio_Interpreting_Plates.shtml
http://delrio.dcccd.edu/jreynolds/microbiology/2421/lab_manual/colony_morph.pdf
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