TITLE

Microbiology

ABSTRACT

The human body has billions of bacteria which constitutes the normal flora fighting against the

invading pathogens. It is tedious to isolate a particular type of bacteria from a clinical sample.

Streak plate technique is used to grow bacteria on a growth media surface so that individual

bacterial colonies are isolated and sampled. Isolated colonies indicate a clone of cells, being

derived from a single precursor cell. When the selected culture media is inoculated using a single

isolated colony, the resulting culture grows from that selected single clone. The modern streak

plate method has evolved from the efforts by Robert Koch and other microbiologists to obtain

pure culture of bacteria in order to study them. The dilution or isolation by streaking procedure

was originally developed by Loeffler and Gaffky in Koch's laboratory, which involves the

dilution of bacteria by systematically streaking them over the surface of the agar in a petri dish to

obtain isolated colonies which will subsequently grow into mass of cells, or isolated colonies. If

the agar surface grows microorganisms which are all the genetically same, the culture is then

considered as a pure culture.

 Staining is an auxiliary technique used in microscopic techniques used to enhance the

clarity of the microscopic image.Stains and dyes are widely used in the scientific field to

highlight the structure of the biological specimens, cells, tissues etc.

 The most widely used staining procedure in microbiology is the Gram stain, discovered

by the Danish scientist and physician Hans Christian Joachim Gram in 1884. Gram staining is a

differential staining technique that differentiates bacteria into two groups: gram-positives and

gram-negatives. The procedure is based on the ability of microorganisms to retain color of the

stains used during the gram stain reaction. Gram-negative bacteria are decolorized by the

alcohol, losing the color of the primary stain, purple. Gram-positive bacteria are not

decolorized by alcohol and will remain as purple. After decolorization step, a counter stain is

used to impart a pink color to the decolorized gram-negative organisms

OBJECTIVES

1. To apply streaking plate method and obtain isolated microbial colonies from an inoculum by

creating areas of increasing dilution on an agar petriplate.

2. To observe bacterial growth by apply those 3 control (using hand sanitizer, water, soap )

with our negative control

3. To apply gram staining method.

APPARATUS AND MATERIAL

1. Agar plates

2. Inoculating loop

3. Bunsen burner

4. 70% alcohol

5. Slide

6. E –coli

7. Staphylococcus aureus

8. Hand sanitizer and soap

9. Safaranin

10. Crystal violet

11. Gram Iodine

METHODOLOGY

PART : STREAKING METHOD

(A) Streak plate technique

1. The inoculating loop was sterilized in the Bunsen burner by putting the loop into the flame until it

is red hot. Allowed it to cool.

2. An isolated colony were chose from the agar plate culture of (a) E. coli and (b) S. aureus and

were spread each of them over the first quadrant on separate agar plate.

3. The agar plate were covered with the lid and flamed the loop.

4. The plate were turned and lightly streaked into the next quadrant without overlapping the

previous streak.

5. Step 3 and 4 were repeated and streaked into the third quadrant.

6. Sealed each plate with parafilm.

7. Inverted the plates and incubated at 37oC for 24 hours.

(B) Effect of hand washing on bacteria on thumb

1. 4 nutrient agar were obtained and labelled them:

a) control

b) water

c) hand sanitizer

d) soap

2. Each agar plate were divided into 4 sections by drawing line using a marker pen on the back of

the petri dish.

3. Aseptic technique were used, gently pressed thumb on the control agar plate.

4. Wash hands (including thumb) with water and repeated step 3 on the appropriate agar.

5. Step 4 repeated using hand sanitizer and soap.

6. Sealed each plate with parafilm.

7. Inverted the plates and incubated at 37oC for 24 hours.

PART : GRAM STAINING

1. Used a sterile inoculating loop, 1 drop of sterile water were added to the slide. A smear of:

(a) Escherichia coli and (b) Staphylococcus aureus were prepared.

2. Air dry and Heat fixed.

3. The smear were covered with Crystal Violet (primary stain) for 1 min.

4. Gently washed off the slide with water.

5. Gram’s Iodine (mordant) were added for 1 min.

6. Washed with water.

7. Decolorized with 95% ethanol. This is the "tricky" step. Decolorizing with alcohol has to stop

as soon as the purple color has stopped leaching off the slide (time will vary depending on

thickness of smear). Immediately wash with water. Be sure to dispose of all ethanol waste in the

appropriately labelled waste container.

8. The smear were cover with Safranin for 30 seconds.

9. Both the top & the bottom of the slide were washed with water.

10. Blotted the slide.

11. Using the light microscope, the smear were viewed up to 100x with immersion oil.
RESULT

Laboratory assistant prepared us with this two different types of bacteria. I chose E-Coli.
Streaking plate method applied onto the plate. This is after the streaking take place. Before the

incubation phase
This is my plate before the observation with the 4 control.

After one day result of streaking plate.

Observation after one day incubation.

Gram staining method applied.

Gram staining observed under microscope . Using species of Escherichia coli
DISCUSSION

Streaking plate method is a common method used to isolate certain colony of bacteria and

culture it in the agar plate with enough nutrients needed for the bacterial growth. The agar plate

contains various of nutrient needed for the bacteria to support it rapid growing. Streaking plate

method also use as an dilution method of bacteria. In this experiment we used pure culture of E-

Coli and dilute it onto other agar plate. The result of the experiment as the picture shown from

the above page. During the 4th streaking on the 4th quadrant were observed to have few colonies

of E-coli bacteria. Rather than first quadrant shown that, rapid growth of bacteria taking place.

Bacteria display a wide variety of nutritional and physical requirements for their growth.

This includes water, a source of energy, sources of carbon, sulfur, nitrogen phosphorus, minerals

such as Ca2+, Mg2+, Na+, and other vitamins and growth factors. Nutrient agar is a complex

medium as it contains ingredients with unknown amounts or types of nutrients. Nutrient agar

typically contains 0.5 % peptone, 0.3 % beef extract, 1.5 % agar in water (pH adjusted to neutral

at 25 °C).

For the next part is, observe the bacterial growth on the agar plate using 4 control stated.

For my plate, it resulting almost no change for bacteria growth in each quadrant. It showed that,
the same growth pattern for the 4 control in each quadrant. I do make a conclusion that, maybe

during the preparation of this control, my techniques applied is wrong from it original procedure.

We do know that microbiology experiment is so sensitive, as it involves with microbes, and our

experiment maybe contaminated with the several of microbes from air, from the table or

anywhere else. Speaking was not allowed and microbes form our clothes may be affected our

result to. This is all factors that could contribute to the failure result. But I do learn during this

experiment to be more extra careful during handling the microbiology experiment.

The Gram stain is a very important preliminary step in the initial characterization and

classification of bacteria. It is also a key procedure in the identification of bacteria based on

staining characteristics, enabling the bacteria to be examined using a light microscope. The

bacteria present in an unstained smear are invisible when viewed using a light microscope. Once

stained, the morphology and arrangement of the bacteria may be observed as well.

The Gram stain procedure enables bacteria to retain color of the stains, based on the differences

in the chemical and physical properties of the cell wall. Gram positive bacteria can stain dark

purple due to retaining the primary dye called Crystal Violet in the cell wall. Staphylococcus

aureus is an example of gram positive bacteria while Escherichia coli is a gram negative

bacteria. Gram negative bacteria can stain red or pink due to retaining the counter staining dye

called Safranin. So our result tally with the theory.

LABORATORY REFLECTION

I learned so many things during the streaking plate method which is quite sensitive to

handle as we did not want those microbes in the air to affected our experiment.Only E-Coli

needed! We may have different colonies of bacteria if those streaking method applied is wrong.

And everything started with sterilization and sterilization. Sterilization is sterilizing media to

above 1210C for 15 minutes in an autoclave destroys nearly all living cells and spores. If screw

caped bottles are used, the cap must be loosened prior to sterilization process. So before we start

the streaking techniques, we must rub all those material and apparatus needed with alcohol and

put it into the chamber. Everything need to handle carefully.

I also failed handling the procedures using those 4 control to observe bacteria growth. We

should get different result but maybe the way conducting the experiment were wrong,

During the gram staining process I learned how to handle the apparatus very well. It is

not easy at first for us as this technique is new to us but as soon demonstrated, I feel excited to

try.Using the microscope to observe quite difficult without the actual procedures. I learned from

my friend and lecturer for the trick using microscope. The key is, patient and keep playing with

the knob.
 REFERENCES

1. Gram staining theory,

https://serc.carleton.edu/microbelife/research_methods/microscopy/gramstain.html

2. Gram staining techniques,

http://vlab.amrita.edu/?sub=3&brch=73&sim=208&cnt=1

3. Streak plate method,

http://vlab.amrita.edu/?sub=3&brch=73&sim=213&cnt=1