Practical 3 - Techniques for staining bacteria Flashcards
Visualisation of living microorganisms is difficult, not only because they are minute, but also because they are…
Transparent and practically colourless when suspended in an aqueous solution
Staining methods can be either
Simple or differential
What is the difference between simple and differential staining?
Simple staining shows you the morphology of the organisms whereas differential staining allows different types of bacteria to be separated
What is the first step in any staining procedure?
To prepare a smear on a slide
When preparing a smear on a slide the aim is to produce a…
Unicellular layer of organisms
Why is a unicellular layer of organisms (thin smear) important when preparing a smear on a slide?
This allows their shape and size to be clearly seen
How is a bacterial smear fixed?
Passed through a bunsen flame three times
Why is it important to fix a bacterial smear onto the slide?
The smear will wash away during the staining procedure otherwise
What does heat fixation serve to do?
To kill the bacteria and cause them to adhere to the surface of the slide - the bacterial proteins coagulate and fix to the surface of the slide
In simple staining, the bacterial smear is stained with a…
single reagent
In simple staining what can be seen?
A distinctive contrast between the organism and its background
Why are basic stains with a positively charged chromogen preferred in simple staining?
The bacterial nucleic acids and certain cell wall components carry a negative charge that strongly attracts and binds to the cationic chromogen
Name the three most commonly used basic stains
- Methylene blue
- Crystal violet
- Carbol fuchsin
Negative staining requires the use of an ______ stain
acidic
Give an example of a stain that can be used for negative staining
Nigrosin
Acidic stains have a ________ charged chromagen
negatively
Why do acidic stains not penetrate the cells of bacterial cells?
The bacterial cells have a negative charge on the bacterial cell surface
In negative staining the unstained bacterial cells are easily seen on a
coloured background
What is an advantage of no heat fixation step in negative staining?
The bacterial cells natural size and shape can be observed
Why should the bacteria be handled with care in negative staining?
They are still alive
Some bacteria are difficult to stain such as some spirilli, why is negative staining ideal here?
Because heat fixation is not carried out
What is the most important staining procedure used in determinative bacteriology?
Gram stain
The Gram stain reaction divides bacteria into
Gram positive and gram negative
The staining reaction in gram stain depends upon the chemical ad physical structure of the bacterial
cell wall
Gram positive bacteria have walls which will retain
crystal violet
Gram negative bacteria cells fail to retain the
crystal violet
What is the primary dye?
Crystal violet
What is the mordant?
Iodine solution
The iodine combines with the crystal violet in the cells to form a compound referred to as…
The crystal violet-iodine complex
What is used as the decolouriser?
Ethanol
Gram negative bacteria are counterstained with…
safranin
Gram positive bacteria should appear purple because they will have retained the
CVI complex during the ethanol treatment
Gran negative bacteria should appear red since they will have been
decolourised by the ethanol and would have taken up the safranin
What method can be used to differentiate between the bacterial spore and vegetative cell forms?
Schaeffer-Fulton method
Give an example of an anaerobic bacteria which is able to form endospores
Clostridium
Give an example of aerobic bacteria which are able to form endospores
Bacillus
What are endospores?
Highly resistant, metabolically inactive cell types, when the environment becomes unfavourable they form and remain to give rise to the metabolically active vegetative cell form when the right conditions return
What is the endospore surrounded by?
Impervious layers called the spore coat
Is heat fixation required in the Schaeffer-fulton method?
Yea babes
What colour will the endospores stain and what colour will the rest of the cell stain?
Endospore - green
rest of cell - pink/red
What is the primary stain used in the Schaeffer fulton method used for staining endospores?
malachite green,
What is the counterstain used in the schaeffer fulton method for staining the rest of the cell?
safranin
