Monday, June 29, 2020
Demonstration of bacterial motility by hanging drop method
Tuesday, June 23, 2020
B Cell Receptor and Antiboby Production
Thursday, June 18, 2020
Sources of Microorganisms in Air
2. Water
3. Plant and Animal Surfaces
4. Human beings
Monday, June 15, 2020
Gram Staining Procedure
Gram stain was introduced in 1880 by the
Danish bacteriologist Christain Gram.
Based on this staining reaction almost all bacteria can be separated
into two large groups called Gram positive and Gram negative.
Aim
To
differentiate between two principal groups of bacteria: gram positive and
gram negative.
Principle
It is a type of differential staining
which uses four reagents and has got four steps. The heat fixed smear of the
organism is first treated with the primary stain called Crystal violet. It is a basic dye and its function is to
impart its color to all cells. At this
stage all the organisms appear in violet color.
In the second step, after washing, the smear is treated with Gram’s
Iodine. This reagent acts as the killing
agent as well as the mordant.
Mordant is a substance that increases the
cell's affinity for a particular stain. It
binds with the primary stain and forms an insoluble crystal violet- iodine
(CV-I) complex. All cells appear purple
black at this stage. In the third step,
the smear is treated with the decolorizing agent, absolute
ethanol. During this process, some bacteria
loose the CV-I complex, whereas some retain the same. In the final step, the smear is treated with
the counter
stain or secondary stain called Safranin. The organisms which have lost the CV-I
complex take up this red dye and appear red in color and are called Gram
negative bacteria. The organisms which
did not lose CV-I complex will not take up the secondary stain and remain
violet in color. They are called Gram positive bacteria.
The difference in the chemical and
physical nature of the bacterial cell wall is responsible for this difference
in response to gram stain. The gram
negative cell wall is thin, complex, multilayered and contains relatively high
lipid contents, in addition to proteins and mucopeptides. The dehydrating agent (absolute alcohol)
readily dissolves the higher amount of lipids leading to the formation of large
pores in the cell wall which do not close appreciably on the dehydration of the
cell wall proteins. Through these pores
the CV-I complex leakage takes place and the cells take up the counter stain. In contrast, the gram positive cell walls are
thick and chemically simple, composed mainly of protein and cross-linked
polypeptides. When treated with alcohol,
it causes dehydration and closure of cell wall pores, thereby preventing the
loss of CV-I complex and cells remain violet colored.
Requirements
24 hr old cultures of Escherichia coli and Staphylococcus spp.
Gram staining reagents:
Crystal
violet
Gram’s
iodine solution
95
% ethyl alcohol
Safranin
Wash bottle of distilled water, staining
tray, droppers, inoculating loop, glass slides, blotting paper, Bunsen burner, microscope
etc.
Procedure
·
On separate slides, make thin smears of
the cultures provided, air dry and heat fix
·
Flood the smear with Crystal violet for
30 sec
·
Wash the slides with distilled water for
few seconds
·
Cover each smear with Gram’s iodine
solution for 30 sec
·
Decolorize the smears by adding ethyl
alcohol drop by drop, until no more color flows from the smear
·
Wash the slides with distilled water and
drain
·
Apply Safranin to smears for 30 sec
·
Wash with distilled water, blot dry with
blotting paper and air dry
·
Observe under oil immersion objective
(100X).
Observations
Escherichia
coli- Rod
shaped (bacilli) cells in chains and appeared red in color
Staphylococcus - Round shaped (cocci) cells in grape like clusters and appeared violet in color.
Result In the given cultures, Staphylococcus spp. belongs to Gram positive bacteria and E. coli to Gram negative bacteria.
Saturday, June 13, 2020
Pure Culture preservation strategies
Short term methods
|
Long term methods
|
·
Agar slant cultures (Sub
culturing) & Refrigeration
|
Mineral
Oil or Liquid Paraffin Method
|
|
Saline
suspension storage
|
|
Drying
in Vacuum
|
|
Storage
at low temperatures (Cryopreservation)
|
|
Lyophilization
(Freeze drying)
|
- Many species remain viable for longer periods under oil than in tubes without oil.
- Sub-culturing can be done as and when required without affecting the stock cultures.
- Useful for strains susceptible to mutations on repeated sub-culturing
- Simple and economical method as equipment like dessicator, vaccum pump etc are not needed.
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