FACULTY OF FOOD SCIENCE AND NUTRITION

NT10502 GENERAL MICROBIOLOGY

PRACTICAL 2: Techniques for Handling Microbial Cultures

GROUP NUMBER:1

DATE (DAY) OF EXPERIMENT:19/11/2018

NAME MATRIX NO. SECTION SIGNATURE

LEE YEK QIUN BN18110044 1
HO QI JING BN18110066 1
NUR ATHIRAH BN18110063 1
HANISAH BT YAAKUB
SYAFAWATI BT AHMAD BN18110058 1
KAMIL
LEE SIOK PEI BN18110109 1
VIVIAN CHONG PIK BN18110048 1
WAN
FELICIA URAI BN18110067 1
BONIFACE
PRACTICAL 3

INTRODUCTION

The purpose of making a smear is to fix the bacteria onto the slide and to prevent the
sample from being lost during a staining procedure. A smear can be prepared from a solid or
broth medium. Heat fixation process is necessary for the preparation of bacterial smear is to
kill the cells so they adhere to the glass and don't get washed away. Bacterial smear is the
first step of the staining process. Staining is a procedure in which a dye or a combination of
dyes and reagents is used to colour the constituents of cells and tissues. This will make the
bacteria visible to our eye after staining. There are two types of staining method, which are
simple staining and differential staining. Simple staining is using a simple basic dye, such as
crystal violet, in order to view the shape , size, and arrangement of cells. Gram staining is
one of the differential staining that are used to characterize bacteria in one of two groups:
either gram positive bacteria or gram negative bacteria. In light microscopy, oil immersion is
a technique used to increase the resolving power of a microscope. This is achieved by
immersing both the objective lens and the specimen in a transparent oil of high refractive
index, thereby increasing the numerical aperture of the objective lens.

OBJECTIVES

1. To know about the techniques of preparing the ideal bacterial smear.

2. To learn about the techniques of handling the compound light microscope to
examine the smear.
3. To know about the techniques and procedures for the both staining which are simple
staining and gram staining.
4. To learn to know the techniques of using oil immersion in microscope.
5. To learn about the cell arrangement, size, shape and colour under microscope.

MATERIALS AND APPARATUS

Ethanol, Glass slide, Inoculum loop, Beaker, Compound Light Microscope, Filter paper,
Bunsen burner, Methylene Blue solution, Distilled water, Oil immersion, Crystal violet
solution, Iodine solution, Safranin, Culture

PROCEDURE

Simple staining

1. The slide was sterilised using the alcohol.

2. A small drop of sterilised distilled water was placed at the centre of slide’s surface
with sterilised inoculating loop.

3. The loop was sterilised, it was allowed to cool, a small amount of culture was picked
out and suspended in the drop of water.

4. The suspension was spread over an area about 1.5cm in diameter. A thin, even
smear was aimed to be obtained. The smear was allowed to dry at room temperature.

5. The bacteria was fixed by passing the slide upwards through the flame 3 times. The
slide should not be overheat.

6. The smear was stained by placing the slide on the rack over the sink and the smear
was covered with staining solution (methylene blue) for 1 minute.

7. The smear was rinsed in tap water with slow running water and filter paper was used
to blot the slide and make it dry. Do not dry the slide by rubbing it.

8. The smear was examined under the oil immersion lens. The shape, arrangement and
colour of the microorganism observed was recorded.

Gram staining

1. A heat-fixed smear from bacterial culture was prepared on a clean slide.

2. The smear was stained with crystal violet solution for 1 minute. The entire smear
should be covered.

3. The crystal violet solution was washed off from the smear with slow running water
and the iodine solution was allowed to act for 1 minute.

4. The iodine was poured off. The slide was washed with alcohol slowly until no more
violet stain runs from the slide. To get a well prepared thin smears, the slide was suggested
to wash in the time of 5 to 15 seconds only to prevent additional decolourization.

5. The slide was rinsed under the tap and stained with safranin for 1 minute.

6. The slide was washed well with slow running water and blotted to dry but do not
rub.

7. The slide was examined with higher power (×40) objective lens under the oil
immersion lens. The morphology was drawn out and the bacterial examined was determined
whether it was gram positive or gram negative.
RESULTS

Simple staining

Total magnification
Charateristics
40x 100x
Shape Coccus Oval
Arrangement “Grape-like” clusters “Grape-like” clusters
Colour Blue Blue

Gram staining

Total magnification: 40x

Total magnification: 100x

Since the colour observed under 40x objective lens and the oil immersion lens is purple, it
shows that the bacterial is gram-positive bacteria.
DISCUSSIONS

1. Why it is necessary to sterile the slide by using the alcohol before preparing
a smear?
The slide may contains the grease or dirt. By sterilizing slide with alcohol can prevent
the bacteria attach to the grease or dirt which may cause them to wash away.

2. What is the purpose of heat fixation in preparation of a smear on a slide?

Heat fixation is used to kill the bacteria and adhere them to the slide so they do not
get washed off throughout the staining process.

3. What problems can arise when the slide is not completely dry while doing
heat fixation?
If heat fixing the wet slide, the water will boiled on the slide and in the cell causing
the shape of the bacteria to be distorted.

4. What will happen if the slide is heated directly in a flame to speed up the
drying process?
This will lead to overheating which will cause the cell wall of the bacteria to be
denatured and ruptured which make lead to over-decolourization.

5. What is the function of each of the following in the Gram’s staining?

a) Simple staining : It is a basic stain that dyes both gram-

(Crystal violet solution) positive bacteria and gram-negative bacteria
on the slide to become purple.

b) Mordant (iodine) : It fixes colour of the primary stain (crystal

violet) into the bacterial cell by binding it to the
RNA to create larger crystal violet-iodine
complex so that it is not removed during the
decolorizing step.
 c) Decolourizer (95% alcohol): It creates large holes in the gram-negative
cell’s outer lipopolysaccharide cell membrane
and washes out the iodine-crystal violet
complex.

d) Counterstain (safranin) :It is used to stain the decolorized

gram-negative bacteria. It gives the
gram-negative bacteria the pink colour. It
also stains the gram positive bacteria, but
because they are already so heavily stained by
crystal violet, the addition of pink does not
change the dark purple colour of the gram-
positive bacteria.

6. Which is the most critical step in the Gram’s staining procedure that is most
likely to cause poor results if done incorrectly?
The most critical step is the decolourizer step as it is the step in which the cells become
differentiated. If too much of the decolourizer is being used, it will lead to over-
decolourization result in the gram positive cells lose the primary stain and be
counterstained pink. While, if it is too little can lead to under-colourization where the
gram negative cells will not lose the primary stain and will remain purple.

7. Why must the thickness of the smear be considered before performing the
Gram’s staining?
If the smear is too thick, there will be a huge clumps of bacteria and the staining will
be uneven. This will make the shape and arrangement of the bacteria difficult to be
observed. It also diminish the amount of light that can pass through making it difficult
to observe the bacteria.

8. For Gram’s staining, how does a counterstain is differ from a primary stain?
A counterstain must have a contrasting colour to the primary stain to aid in
differentiation.
9. Why must fresh bacterial cultures (within 24 to 48 hours) be used in a
Gram’s stain?
Only fresh bacterial cultures should be used in a Gram stain. It is to avoid any improper
gram-staining characteristics due to the cell wall integrity of older cells. Gram-positive
bacteria that have lost cell wall integrity because of old age may appear pink.

10. How does smear preparation of cells from an agar media differ from
preparation of cells from a broth media?
To prepare smear from solid media, bacteria must be mixed in one drop of water on
the slide. While for the broth media, one or two loopfuls of liquid suspension can be
transferred directly to the slide.

11. Predict what will happen if the smeared is counterstained for more than 1
minute?

Overcounterstaining result in the leaching of the crystal violet-iodine complex from

gram-positive bacteria and stain them with safranin, thus making them falsely appear
as gram-negative bacteria.

12. What is the difference between a simple and a gram’s staining?

Simple staining Gram’s staining

It only involving one stain, either It involves two type of stain which are
methylene blue, Gram safranin or Gram primary stain (crystal violet solution) and
crystal violet. counterstain (safranin).

It is used to colour the cell of bacteria It is used to distinguish between gram-

which is invisible to determine their positive and gram-negative bacteria.
morphology such as shape, size and
arrangement.

13. Why are Gram-positive bacteria stain purple and Gram-negative bacteria
stain pink in colour?

Gram-positive bacteria stains purple because they contains the thick layer of
peptidoglycan, high numbers of cross-linked teichoic acids, and little lipid. This makes
 them less permeable to organic solvents (alcohol in this case). As a result, the primary
dye does not wash out during the decolourization step. The cell walls of gram-negative
bacteria, on the other hand, have a thinner peptidoglycan layer and a
lipopolysaccharide outer cell membrane which increase the permeability to organic
solvents. This allows the primary stain to wash out during the decolourization step.
The cell walls then take up the counterstain and the bacteria appear pink.

14. Why it is important to use immersion oil with the 100x objective lens?
Immersion oil with a refractive index equal to that of the glass light in the space filled
with air helps to minimize the light scatter so more light is directed through the
objective lens and a clearer image can be observed.

CONCLUSION

An ideal bacterial smear should be thin, semi-transparent, whitish layer or film, circular with
diameter 1 cm(approximately), free from dirt, dust or any contamination. By using oil
immersion in microscope with 100X objective lens, the image observed is higher
magnification greater clearness. For the simple staining, the bacteria is oval shape. For the
gram staining, the bacteria is in purple colour which mean that the bacteria are Gram-
positive. This is because the bacteria contains the thick layer of peptidoglycan, high
numbers of cross-linked teichoic acids, and little lipid. This makes them less permeable to
organic solvents.

REFERENCES

Alfred E. Brown. 2009. BENSON’S MICROBIOLOGICAL APPLICATIONS: LABORATORY

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