Microbiology lab prac revision

Cellular morphology of bacteria:

1 Shape:
Cocci have a spherical shape.
Bacillus have a rod shape
2 Gram Positive or Gram negative stains:
Gram Positive bacteria stain purple and they contain a thick
peptidoglycan layer and an cell membrane layer.
Gram negative bacteria stain pink and they contain two plasma
membrane and adjoined to the top plasma membrane is a thin
peptidoglycan layer.
3 Arrangement
The bacteria can be arranged in pairs, chains, clusters or randomly.
4 Spores
Some bacteria may have protective spores (rounded resistant
structures formed when the bacteria is facing adverse conditions)

The Streak plate uses on agar plate to try and create on isolated
colony.
Other techniques include: Spread plate and Pour plate
Aseptic conditions: conditions that prevent contamination (Bunsen
burner)

Agar in General
Agar is a complex polysaccharide that scientists derive from marine alga.
It possesses several unique properties that make it valuable to
microbiologists. First, few microbes can degrade agar, so it remains solid.
Second, it will not liquefy until it reaches a temperature of 100 Celsius,
and once liquefied it will remain so until brought down to 40 Celsius. Its
ability to remain solid at high temperatures make it an ideal medium for
growing thermophilic (heat-loving) bacteria.
Nutrient Agar
Since agar is only a solidifying agent, it carries no value for the bacteria
grown on it. Bacteria need nutrients to live and reproduce. One solution
involves the mixing of agar with a nutrient broth, containing peptone and
beef extract, to create nutrient agar.
Nutrient Agar Is a Complex Media.
For practical purposes, nutrient agar works well for growing most types of
non-fastidious heterotrophic bacteria. "Fastidious" means selective, and
"heterotrophic" means the bacteria cannot make their own food. Nonfastidious heterotrophic bacteria, therefore, need their food supplied to
them, and they are not fussy about from where it comes. Since many
pathogenic (disease-causing) bacteria fall into the non-fastidious

heterotrophic category, a complex media consisting of various nutrients

such as peptones and beef extracts is the ideal choice for bacterial growth
and cultivation.
Blood Agar
Blood agar is almost identical to nutrient agar except that it contains five
to ten percent sheep, rabbit, or horse blood. Blood agar consists of beef
extract, for nitrogen, blood, for nitrogen, amino acids and carbon, sodium
chloride, for maintaining osmotic balance, and agar. Microbiologists use
blood agar to identify fastidious pathogenic bacteria by studying the
hemolytic (blood cell destroying) reactions they cause.
Blood Agar Is a Differential Media
Microbiologist use differential media to identify and isolate specific
bacteria. An example of this is the bacteria Streptococcus pyogenes,
which causes strep throat. You can grow these bacteria on a complex
media such as nutrient agar, but if other bacteria are also growing on that
agar, it is very difficult to distinguish one bacterial colony from another
without the use of microscopic examination and special staining
techniques. If you grow it on blood agar, though, it will destroy the red
blood cells in a process called beta-hemolysis, and other cells will not
cause this reaction, which makes identifying Streptococcus pyogenes
much easier.
Stains
Crystal violet
Iodine Binds to
crystal violet complex
(C-VI complex),
making a bigger
complex structure to
keep the stain in the
bacteria cell.
Ethanol

Carbol Fusion (pink

stain)

Gram Positive
Stains Gram
Positive (purple)
Still stains purple

Gram Negative
Stains Gram
Positive (purple)
Still stains purple

Ethanol does not do

this to the Gram
Positive cell. It
remains purple

Ethanol creates
holes in the Gram
negative bacteria.
The C-VI complex
comes out and
leaves the cell. The
Gram negative
bacteria at this
stage is colourless.
The carbol fusion
(pink stain) enters
the cell and the
Gram negative
bacteria remains

The pink stain

enters the Gram
Positive cell but it
remains purple, as
purple is the darker

colour.

pink.

If you forgot to administer Crystal violet Both Gram Positive

and Gram negative stain pink.
If you forgot to administer Iodine Both Gram Positive and
Gram negative would stain pink.
If you forgot to administer ethanol Both Gram Positive and
Gram negative would stain purple.

Sample A: Bacillus ceres does not cause respiratory infection

Sputum sample: mixed culture many flora live in our saliva naturally,
many bacterial species.
Pure culture one type of bacterial colony.
Sample B: S.pyrogenes
- Causes tonisiltis and respiratory infections.
Bacillus anthrax: contains spores and causes bad respiratory infection.