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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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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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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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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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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

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.)

Per Bench

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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.