CHAPTER 2:

MICROBIAL STRUCTURE
SPECIMEN PREPARATION
GENERAL MICROBIOLOGY MIC461 | NMJ
LEARNING OUTCOMES
At the end of this topic, students should be able to:

• Understand the concept of microscopy

• Differentiate the different types of microscopes
• Describe sample preparation strategies
• Explain the process and theory behind staining
techniques in microbiology
Light Microscope Specimen
Preparation
• Wet Mounts
• Smears
• Placement of cells
• Air drying
• Heat fixation

Copyright © 2018 by John Wiley & Sons, Inc.

All rights reserved.
 Light Microscope Specimen
Preparation
▪ Smear preparation
• Smear is a preparation of a thin film or layer of sample over
a solid surface eg. microscope slide
• There are several types of smear –
• blood smear - thin blood smear or thick blood smear
• bacterial smear
• buccal swab/smear
• Correct techniques in the preparation of smear is crucial in
obtaining good image in microscopy

Copyright © 2018 by John Wiley & Sons, Inc.

All rights reserved.
Thin blood smear
Thick blood smear

https://goo.gl/images/4NxQ7m

Copyright © 2018 by John Wiley & Sons, Inc.

All rights reserved.
 Buccal swab/smear
• Samples are taken using sterile swab from
mucosal site – oral, vaginal or anal

https://goo.gl/images/tRXLhm https://goo.gl/images/7YA26R
Bacterial smear
◼ Smear can either be made from liquid or solid media
◼ If from solid media, a drop of sterile saline is usually need in order to get
an evenly spread smear.
◼ Often bacterial smear require fixing in order to
– kill bacteria
– ensure sample adheres to the glass slide
– preserve and minimize distortion of cells
◼ Two types of fixing
– Heat fixing – running of the slide with air-dried smear on top of a
flame
– Chemical fixing – applying methanol over air-dried smear for about 1
minute
 Bacterial smear
Steps of microbial smear preparation:
• Sterile the loop until reaching the red heat .
• If the bacterial culture was broth, shake the
culture and transfer loopful of broth to the
center of the slide and spread over the target
circle .
• While if the bacteria were grown on solid
medium, place loopful of water on the slide
then transfer inoculums to the water and
homogenize the smear.
• Leave the smear to dry at room temperature
(by air).
• After drying, pass the slide over the flame to
fix the smear (avoid prolonged heating of the
slide).
Specimens preparations
1
Because most microorganisms appear almost colorless when
viewed through a standard light microscope.

2
One way to do this is to stain 3
the specimen. Before the microorganisms can be stained,
however they must be fixed (attached)to the
microscope slide.

5 4
◼ A thin film of material Fixing simultaneously kills and fixes them to
containing the the slide
microorganisms is spread over
the surface of the slide.
◼ This film, called a SMEAR is
allowed to air dry.
Copyright © 2018 by John Wiley & Sons, Inc.
All rights reserved.
Function of staining
– Once smears has been prepared, the next step would be
staining
– Function of staining
• Allow cells to be seen more easily
• Differentiate the types of cells
• Observe certain structures of the cell
• Differentiate different status of cells
◼ Stains or dyes are generally salts of positive or
negative ions called chromophores

ACIDIC DYES BASIC DYES

• negatively charged (anions) • positively charged (cation)
• Often used for staining of • attracted to any negatively
background (negative staining) charged cell components.
• Bacteria are not readily stained • Bacteria are readily stained by
by acidic dyes – bacterial cell basic dyes – bacterial cell wall are
wall are slightly negatively slightly negatively charge
charge Examples : Crystal violet, Methylene
• used to in capsule staining blue, Malachite green, Safranin
Examples : Eosin, Acid fuchsin,
Nigrosin
Capsule staining

https://goo.gl/images/sbYrak
Simple Staining
• Simple staining
• Used generally to observe the morphology and appearance of
bacteria
• do not distinguish organisms or structures by different staining
reactions
• Make use of only one dye - Methylene blue, Safranin, Crystal
violet
• Procedure:
• Stain is allowed to come in contact with prepared bacterial smear
for a certain amount of time
• The stain is then washed off with water and the stained smear is
allowed to air-dry before cells are observed under microscope
• Mordant is some times used to intensify the stain color
Differential Staining – Gram Staining
◼ Differential staining - Gram staining
– Was first developed by Hans Christian Gram in 1884 – thus the “G”
in Gram has to be capitalized since it’s a person’s name
– Make use of two dyes, one mordant and one decolorizor
– Frequently used in microbiology especially medical microbiology
– Differentiates between Gram positive and Gram negative bacteria
– Drawback:
• Cannot stain acid fast bacteria
• Works well on young culture (16-18 hr). Old cultures of Gram
positive bacteria results in Gram variable
Gram Staining Procedure

https://goo.gl/images/D9LkCQ
https://goo.gl/images/54Ww
TG
Copyright © 2018 by John Wiley & Sons, Inc.
All rights reserved.
Gram Staining Procedure

• Technique
• Gram +
• Gram –
• Significance
• Cell wall anatomy
• Diagnosis

Copyright © 2018 by John Wiley & Sons, Inc.

All rights reserved.
Differential Staining – Acid fast staining
◼ Differential staining – Acid fast staining
– Also known as Ziehl-Neelsen staining
– Use to stain acid fast bacteria (Mycobacteria)
• Contains large amount of lipids in their cell wall
• Normal basic dye cannot be readily absorbed
– Make use of carbol fuchsin, acid alcohol and methylene
blue
• Acid fast bacteria will appear bright red against blue
background
Acid fast Staining Procedure

https://goo.gl/images/3UDp9S
https://goo.gl/images/gCpmKa
Ziehl-Neelsen Acid Fast
Stain
• Acid Fast Bacteria
• Mycobacterium

Copyright © 2018 by John Wiley & Sons, Inc.

All rights reserved.
 Differential Staining – Capsule staining
• Special staining – Capsule staining
• Capsule is a semi-rigid polysaccharide layer produced by some bacteria as
protection against
• Phagocytosis – virulence factor
• Dryness
• Chemicals – such as detergent
• Viruses
• Since capsule does not readily retain dyes, negative staining methods is
often used whereby the cells are stained with safranin or crystal violet
(basic dye) prior to flooding the smear with Indian ink to stain the
background
• Capsules appear as a halo ring around the red-stained dye/ purple-
stained dye
 Capsule Staining Procedure
1. Place a drop of Indian ink on one end of your slide.
2. Then add your bacteria to the Indian ink and make a smear (like a
blood smear)
3. Air-dry the smear BUT DO NOT HEAT FIX
4. Flood the smear with safranin for 30 seconds to 1 minute – be gentle
because the smear has not been heat-fixed
5. Rinse the stained smear and allow the slide to air-dry.
6. Observe under a microscope.
Capsule staining
• Negative staining
• Stains background, not organism

Copyright © 2018 by John Wiley & Sons, Inc.

All rights reserved.
Copyright © 2018 by John Wiley & Sons, Inc.
All rights reserved.
Special Staining – Endospore staining
• Special staining – Endospore staining
• Schaeffer-Fulton stain
• After staining, bacterial cell will appear red (due to safranin) while the
endospore will appear green (due to malachite green)

https://goo.gl/images/GX2HGx https://goo.gl/images/1C7we3
Special Staining – Endospore staining
• Heat-resistant
endospores
• Schaeffer-Fulton
stain
• Medical
significance

Copyright © 2018 by John Wiley & Sons, Inc.

All rights reserved.
Special Staining –
Flagellar staining Endospore staining
• Special staining – Flagella staining
• Flagella are long filamentous appendages that some bacteria
possess to facilitate motility
• They are generally too thin to be seen under the microscope
• Staining procedures are often difficult and usually require mordant
and coating of the flagella surface with metal or dyes
• In medical microbiology, the number and arrangement of flagella
can help in making diagnosis

Copyright © 2018 by John Wiley & Sons, Inc.

All rights reserved.
 https://goo.gl/images/SCqJ7u

Copyright © 2018 by John Wiley & Sons, Inc.

All rights reserved.
Special Staining – Endospore staining

https://goo.gl/images/3f8Rqn https://goo.gl/images/aTR9Qh
Copyright © 2018 by John Wiley & Sons, Inc.
All rights reserved.
 ANSWER ME NOW….

1. State the purpose of heat fixation in the preparation of a bacterial

stain. (3 marks)

Answer :

Heat fixation kills the microbes and causes the specimen to adhere to the
glass slide so that it does not easily wash off during staining. Fixation
generally also preserve the shape and size of the microbes.
 ANSWER ME NOW….

2. Explain the relationship between contrast and staining in microscopy.

(4 marks)

Answer :

Contrast refer to differences in intensity between two objects or between an

objects and its background and important in determining resolution because
microbes are colorless.

Stain are used to make microbes and their parts more visible because satins
increase contrast between structures and between specimen and its
background.
Learning outcomes
At the end of this topic, students should be able to

– Explain and use the metric system

– Understand the concept of microscopy
– Differentiate the different types of microscopes
– Describe sample preparation strategies
– Explain the process and theory behind staining
techniques in microbiology

Copyright © 2018 by John Wiley & Sons, Inc. All rights reserved.