1. Introduction to Basic Bacteriology
TRANSFERRING OF BACTERIAL COLONIES USING ASEPTIC TECHNIQUE.
INTRODUCTION TO BACTERIAL STAINING
Introduction
Aseptic Technique
A strictly sterile (aseptic) procedure is essential when collecting the specimen from the sites
that are normally sterile such as collection of blood, cerebrospinal fluid (CSF), or effusions.
An aseptic technique is used not only to prevent contamination of the specimen but also to
protect the patient and worker.
Microbiologists and students of microbiology may use an aseptic technique to attempt to
keep specimens of microorganisms free of microbial contamination. People may commonly
use the aseptic technique when transferring bacteria from one test tube or flask to another.
This method of preventing unwanted microorganisms from gaining access is termed as
ASEPTIC TECHNIQUE.
Inoculating Loops (Bacteriological Loops) and Needles
The inoculating loop is sterilized by passing it through the flame of a gas burner at an angle
until the entire length of the wire becomes orange due to the heat. In this way all
contaminants on the wire are incinerated. It is imperative that you can incinerate the entire
wire to ensure absolute sterilization. The shaft should also be briefly passed through the
flame to remove any dust or possible contaminants. To avoid killing the cells and splattering
the culture, cool the inoculating wire by tapping the inner surface of the culture tube or the
edge of uninoculated agar or medium prior to obtaining the inoculum.
Preparation of Smear
Avoidance of thick, dense smears is absolutely essential. A good smear is one that, when
dried, appears as a thin translucent whitish layer or film. Those made from broth cultures or
colonies from a solid medium require a variation in technique.
Broth Culture:
One or two loopfuls of suspended cells should be applied directly to the centre of a clean
glass slide with a sterile inoculating loop and spread evenly over an area about the size of a
10 cent coin.
Allow this thin smear to completely air dry.
Cultures from a solid medium:
Aseptically remove a small amount of the culture from the agar surface with a sterile loop
and just touch it several times to a drop of sterile water on a slide until it just turns cloudy.
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2. Burn the remaining bacteria off of the loop. (If too much growth is emulsified the smear will
be so dense that you won’t be able to see stained individual bacterial cells).
Using the loop, spread the suspension to form a thin smear about a 10 cent coin in size.
Allow this thin smear to completely air dry.
Heat Fixation
Before staining bacteria, you must first understand how to "fix" the organisms to the glass
slide. If the preparation is not fixed, the organisms will be washed off the slide during
staining. A simple method is that of air drying and heat fixing. The organisms are heat fixed
by rapid passage of the air-dried smear two to three times over the flame of a Bunsen burner.
The heat coagulates the organisms proteins causing the bacteria to stick to the slide.
Caution: Too much heat might distort the organism and, in the case of the Gram stain, may
cause gram-positive organisms to stain gram-negatively. The slide should feel very warm but
not too hot to hold.
Staining
Since bacteria are almost colorless and therefore show little contrast with the broth in which
they are suspended, they are difficult to observe when unstained. Staining microorganisms
enables one to:
see greater contrast between the organism and the background,
differentiate various morphological types (by shape, arrangement, gram reaction,
etc.),
observe certain structures (flagella, capsules, endospores, etc.).
In order to understand how staining works, it will be helpful to know a little about the
physical and chemical nature of stains. Stains are generally salts in which one of the ions is
colored. (A salt is a compound composed of a positively charged ion and a negatively
charged ion.) For example, the dye methylene blue is actually the salt methylene blue
chloride which will dissociate in water into a positively charged methylene blue ion which is
blue in color and a negatively charged chloride ion which is colorless.
Dyes or stains may be divided into two groups: basic and acidic. If the color portion of the
dye resides in the positive ion, as in the above case, it is called a basic dye (examples:
methylene blue, crystal violet, safranin). If the color portion is in the negatively charged ion,
it is called an acidic dye (examples: nigrosin, congo red).
Because of their chemical nature, the cytoplasm of all bacterial cells have a slight negative
charge when growing in a medium of near neutral pH. Therefore, when using a basic dye,
the positively charged color portion of the stain combines with the negatively charged
bacterial cytoplasm (opposite charges attract) and the organism becomes directly stained. An
acidic dye, due to its chemical nature, reacts differently. Since the color portion of the dye is
on the negative ion, it will not readily combine with the negatively charged bacterial
cytoplasm (like charges repel). Instead, it forms a deposit around the organism, leaving the
organism itself colorless. Since the organism is seen indirectly, this type of staining is called
indirect or negative, and is used to get a more accurate view of bacterial size, shapes, and
arrangements.
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3. Streaking
Streak the loop across the surface of the agar medium to obtain single isolated bacterial
colonies originating from a single bacterium or arrangement of bacteria. In order to avoid
digging into the agar as you streak the loop over the top of the agar you must keep the loop
parallel to the agar surface. Always start streaking at the "12:00 clock position" of the plate
and streak side-to-side as you pull the loop toward you. As you follow, each time you flame
and cool the loop between sectors, rotate the plate counterclockwise so you are always
working in the "12:00 clock position" of the plate. This keeps the inoculating loop parallel
with the agar surface and helps prevent the loop from digging into the agar.
EXPERIMENT 1: Transferring Of Bacterial Colonies Using Aseptic Technique
In the future, every procedure in the lab will be done using these similar aseptic techniques.
This procedure for aseptically transferring microorganisms is as follows:
1.1 Sterilize the inoculating loop.
The inoculating loop is sterilized by passing it at an angle through the flame of a gas
burner until the entire length of the wire becomes orange from the heat. In this way all
contaminants on the wire are incinerated. Never lay the loop down once it is sterilized
or it may again become contaminated. Allow the loop to cool a few seconds to avoid
killing the inoculums.
1.2 Remove the inoculums. (to be done in pair)
You are provided with organisms Staphylococcus epidermidis (labeled A) and
Klebsiella pneumoniae (labeled B) growth on a nutrient agar (NA) plate.
A. Transferring the inoculum from a plate dish (organisms growing on an agar
surface in a Petri plate) into a broth tube
Sterilize the inoculating loop in the flame of a gas burner until it is orange.
Allow the loop to cool a few seconds
Lift the lid of the culture plate slightly. Scrape off a small amount of the
organisms labeled A.
Pick up a sterile nutrient broth tube and remove the cap with the little finger of
your loop hand. Do not set the cap down.
Briefly flame the lip of the broth tube
Place the loopful of inoculum into the broth, and withdraw the loop. Do not lay
the loop down on the bench!
Again flame the lip of the tube. Replace the cap
Repeat the above procedure for the organism labeled B.
Resterilize the loop by placing it in the flame until it is orange. Now you may
lay the loop down until it is needed again.
Incubate the broth in 37°C incubator for 1 hour.
After 1 hour incubation, use this broth for the Experiment 1.2B
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4. B. Transferring inoculum from a broth culture (organisms growing in a liquid medium)
to the solid medium surface (Nutrient Agar) and Streaking
Labeled your broth tube as A or B. Use this broth later for the experiment 2.2
Hold the culture tube in one hand and in your other hand, hold the sterilized
inoculating loop
Remove the cap of the pure culture tube with the little finger of your loop hand.
Never lay the cap down or it may become contaminated.
Very briefly flame the lip of the culture tube. This creates a convection current
which forces air out of the tube and preventing airborne contaminants from
entering the tube.
Keeping the culture tube at an angle, insert the inoculating loop and remove a
loopful of inoculum.
Again flame the lip of the culture tube. Replace the cap
Transfer the inoculum to the agar surface. Proceed with streaking as the
instruction given below
Lift the edge of the lid just enough to insert the loop. Streak the loop across the
surface of the agar medium.
The streaking allows you to obtain single isolated bacterial colonies originating
from a single bacterium or arrangement of bacteria
Resterilize the inoculating loop
* In the future, every procedure in the lab will be done using similar aseptic
technique
EXPERIMENT 2: STAINING (to be done individually)
Prepare two smears on two different slides of the normal flora and cells of your mouth. Use
one for simple staining and another one for the differential staining.
Procedure
Using a sterile cotton swab, vigorously scrape the inside of your mouth and gums.
Rub the swab over the slide ( do not use water)
2.1 Simple Staining
a. Heat-fix the smear
b. Stain with crystal violet for one minute.
c. Wash off the excess crystal violet with water.
d. Blot dry and observe using oil immersion microscopy.
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5. 2.2 Differential Staining
Do smears on different slides and proceed with Gram staining
Student 1 – From plate labeled A
Student 2 – From plate labeled B
Per Bench
Student 3 – From incubated nutrient broth labeled A
Student 4 – From incubated nutrient broth labeled B
Gram Stain: The most important stain used in bacteriology,
Procedure
Air dry and heat fix.
Flood the fixed smear with crystal violet for 1 min. Wash off the stain with tap
water.
Flood the smear with Lugol’s iodine for 1 min. Then wash off with tap water.
Decolourize rapidly with iodine-acetone and immediately wash with tap water.
Lastly flood the smear with dilute carbol fuchsin for 1 min. Then wash with
water.
Wipe the back of the slide, and place the smear on the bench to air dry.
Examine the smear microscopically, first with the 10X objective to see the
distribution of smear and then examine the smear with oil immersion by using
the 100X objective.
Result: the stain divides bacteria into 2 groups
Gram positive bacteria stain a purple colour
Gram negative bacteria are stained red
CAVEATS
Remove any immersion oil from the oil immersion lens (x100 Objective) before
putting the microscope away using lens paper. (Paper towel or kim-wipes may
scratch the lens.)
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6. DEMO SLIDE
2.3 Negative Staining
In negative staining, the negatively charged color portion of the acidic dye is repelled by the
negatively charged bacterial cell. Therefore the background will be stained and the cell will
remain colorless.
Procedure
a) Place a drop of Indian Ink toward one end of the slide.
b) Aseptically place a loopful of the inoculum of Klebsiella pneumoniae into the
drop of stain and mix with the loop
c) Place a slide against the drop of suspended organisms and allow the drop to spread
along the edge of the applied slide
d) Push the slide away from the previously spread drop of suspended organisms,
forming a thin layer.
e) Air dry the smear and then observe using oil immersion microscopy.
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