ERT 103/4: ENGINEERING MICROBIOLOGY
TUTORIAL 1 – MICROSCOPY TECHNIQUES
1. Assume that a smear has been made on a slide and heat fixed, and the smear
contains both Gram-positive and Gram-negative colourless bacteria. In detail,
describe the staining procedure by using the following terms; primary stain,
mordant, decolorizing agent and counter stain.
i)
Flood the smear with the basic dye crystal violet for 1 min, and then
rinse with water. Crystal violet, which is called the primary stain,
colour all cells.
ii)
Flood the smear with an iodine solution for 1 min, and then rinse with
water. Iodine is a mordant, a substance that binds to a dye and makes it
less soluble. After this step, all cells remain purple.
iii)
Rinse the smear with a solution of ethanol and acetone for 1030seconds, and then rinse with water. This solution, which acts as a
decolorizing agent, breaks down the thin cell wall of Gram-negative
cells allowing the stain and mordant to be wash away. These cells are
now colourless. Gram-positive cells, with their thicker cell walls
remain purple.
iv)
Flood the smear with safranin for 1 minute and then rinse with water.
This red counterstains provides a contrasting colour to the primary
stain. Although all types of cells may absorb safranin, the resulting
pink colour is masked by the darker purple dye already in Grampositive cells. After this step, Gram-negative cells now appear pink,
whereas Gram-positive cells remain purple.
2. In what ways are the Gram stain and the acid-fast procedures similar? In the acidfast procedure what take the place of Gram’s iodine mordant?
Both the Gram and the acid-fast staining procedures differentiate cells on the
basis of different cell wall structures (thick versus thin peptidoglycan in Gram
stains, waxy versus non-waxy in the acid-fast stain), and both procedures use a
decolorizing step to remove the initial dye from cells in which it has not been
trapped, followed by a second dye to make the decolorized cell visible. Heating
followed by cooling in the acid-fast procedure takes the place of the Gram’s
iodine mordant: Heating allows the dye to penetrate the cell wall; cooling traps
the dye inside the cell wall.
3. In detail, describe the procedure in preparing a specimen for staining. Why must a
smear be fixed to the slaid?
1) Making a smear and fixing it to the slide
-
If the organisms are growing in a liquid, a small drop is spread across the
surface of the slide.
-
If the organisms are growing on a solid surface such as an agar plate they are
mixed into a small drop of water on the slide.
2) The smear is air dried and then attach or fixed to the surface of the slide
-
In heat fixation, the slide is gently heated by passing the slide, smear up.
Through the flame of a Bunsen burner.
-
In chemical fixation, applying methyl alcohol (methanol) or formalin (a
solution of formaldehyde in water) to the smear for 1 minutes.
Desiccation (drying) and fixation kill the microorganism, attach them firmly to
the slide and generally preserve their shape and size. It is important to smear and
fix specimens properly so that they are not lost during staining. [2 marks]
4. Explain how stains used for electron microscopy differ from those used for light
microscopy.
To increase contras and resolution for electron microscopy we use stains, just as
for light microscopy. However, stains used for electron microscopy are not
colored dyes, but instead chemicals containing atoms of heavy metals such as
lead, osmium, tungsten and uranium, which absorb electrons. Electron-dense
staining may bind to molecules within specimens or they may stain the
background. Stains for electron microscopy can be general, in that they stain most
objects to some degree, or they may be highly specific.
5. Compare and contras Streptococcus pyogenes with Mycobacterium tuberculosis
which are appropriately stained.
Streptococcus pyogenes – gram stain
Mycobacterium tuberculosis – acid fast stain
Both the Gram and the acid-fast staining procedures differentiate cells on the
basis of different cell wall structures (thick versus thin peptidoglycan in Gram
stains, waxy versus non-waxy in the acid-fast stain), and both procedures use a
decolorizing step to remove the initial dye from cells in which it has not been
trapped, followed by a second dye to make the decolorized cell visible. Heating
followed by cooling in the acid-fast procedure takes the place of the Gram’s
iodine mordant: Heating allows the dye to penetrate the cell wall; cooling traps
the dye inside the cell wall.
6. Why do basic dyes stain bacterial cells? Why don’t acidic dyes stain bacterial
cells?
Bacteria cells have a slightly negative charge, and the colored positive ion of a
basic dye is attracted to the negative charge of the cell. Acid dyes do not stain
bacterial cells because the negatively charged colored ion is repelled by the like
charge of the cell.
7. Why is a mordant used in the Gram stain? In the flagella stain?
In gram stain, the mordant combine with the basic dye to form a complex that will
not wash out of gram-positive cells, in a flagella stain, the mordant accumulates
on the flagella so that they can be seen with a light microscope.
8. Why isn’t the Gram stain used on acid-fast bacteria? If you did Gram stain acidfast bacteria, what would their Gram reaction be? What is the Gram reaction of
non-acid-fast bacteria?
The high lipid content of acid-fast cell walls makes them impermeable to most
stains. If the primary stain penetrates, the Gram stain decolorizer will not
decolorize the cell. Therefore, acid-fast bacteria would be gram-positive if they
could be Gram stained.
9. Endospore can be seen as refractile structures in unstained cells and as colorless
areas in Gram-stained cells. Why is it necessary to do endospore stain to verify
the presence of endospores?
Inclusions as well as endospores may not stain in a Gram stain. The endospore
stain will identify the unstained structure as an endospore.
10. Answer the following question using the diagrams provided, which represent
cross section of bacterial cell walls.
Teichoic acid
Lipopolysaccharide
Phospholipid
Lipoprotein
Peptidoglycan
Peptidoglycan
Plasma membrane
Plasma membrane
(a)
i)
(b)
Which diagram represents a gram-positive bacterium? Why?
Diagram (a) refers to a gram-positive bacterium because the
lipopolysaccharide-phospholipid-lipoprotein layer is absent.
ii)
Explain how the Gram stain works to distinguish between these two types
of cell walls.
The Gram-negative bacterium initially retains the violet stain, but it is
released when the outer membrane is dissolved by the decolorizing agent.
After the dye-iodine complex enters, it becomes trapped by the
peptidoglycan of Gram-positive cells.
iii)
Why does penicillin have no effect on most gram-negative cells?
The outer layer of the Gram-negative cells prevents penicillinfrom
entering the cells.
iv)
How do essential molecules enter cells through each wall?
Essential molecules diffuse through the Gram-positive wall. Porins and
specific channel proteins in the Gram-negative outer membrane allow
passage of small water-soluble molecules.
v)
Which cell wall is toxic to human?
Gram-negative
vi)
Name a bacterium for each diagram.
(a) E. coli (b) B. aureus