2. The streaking is process by which microorganisms
are spread on the surface of the agar medium by an
inoculating loop making streak marks. The
bacterial colonies are produced along the streak
mark. The plate which is prepared by streaking is
called streak plate. Streak plate method is used for
obtaining pure culture from mixed population.
Streak plate gives well separated and isolated
colonies of bacteria which can easily be isolated
preserved as pure culture. This method can also be
used for the study of various characteristics of
bacteria.
4. Preparation of an agar plate
Sterilization of an inoculating loop by read heat
Cooling of the loop by jabbing in the agar plate
Small amount of bacterial sample inoculated on
the agar surface by marking parallel streak marks
Loop sterilizes again and cooled and streaking
continued from the end point of previous streak
mark
This process repeated several times
Incubate the streak plates at 350C for 24 to 48 hrs
5. Bacterial growth could be observed along the
streak marks and separated colonies on the
later streak marks
6. An agar plate streaked with
microorganisms
isolated from a deep-water
sponge. Individual
colonies may be seen center
right.
7.
8. Streaking is a technique used in microbiology to isolate
a pure strain from a single species of microorganism, often
bacteria. A microbiological culture can be grown so that
the organism can be identified, studied, or tested. A sterile
cotton swab or inoculation loop is sterilized and dipped in
a broth or patient specimen containing many species of
bacteria.
9. The loop is then spread across one quadrant of an
agar plate containing a growth medium which has
been sterilized in an autoclave. This introduces a
solution of the bacteria or fungi to a substrate
which provides them nutrients. Choice of which
growth medium to use depends on which
microorganism is being cultured, and which are
being selected for, if any. Growth media are
usually based on agar, a gelatinous substance
derived from seaweed.
10. The loop is re-sterilized and dragged across the
inoculated quadrant of the streak plate. This is
done to collect some bacteria on the loop. The loop
is spread around another fourth of the plate much
like the previous step. The loop is sterilized and
the procedure is repeated. Each time the loop
gathers fewer and fewer bacteria until it gathers
just one single bacterial cell that can grow into a
colony.
11. The streak plate is then incubated, usually for 24 to
36 hours, to allow the bacteria to reproduce. At the
end of incubation there should be enough bacteria
to form visible colonies in the areas touched by the
inoculation loop. From these mixed colonies,
single bacterial or fungal species can be identified
based on their morphological (size/shape/colour)
differences, and then sub-cultured to a new media
plate to yield a pure culture for further analysis.
13. Nutrient broth medium contain sufficient nutrient
and favorable environment for the growth of
majority of bacteria. Nutrient broth is suitable for
the growth of aerobic, anaerobic, facultative,
oxidative and fermentative bacteria. Broth culture
is used for the enrichment and study of many
biochemical characteristics of bacteria.
15. Preparation of broth tube (transfer of 5.0 ml
nutrient broth by micro-pipette)
Inoculation of broth tube with bacterial sample
Incubation at 350 C until next class
16. Bacterial growth was indicated by-
Cloudiness of the medium
Pellicle or firm formation on the surface of the
medium
Sediment formation on the bottom of the tube
17. Culture of Bacteria in an Agar Plate by
Pour Plate Method
18. This is a method by which bacteria are cultured
throughout the agar medium. Although the
solidified agar medium restricts the movement of
bacteria, the medium is soft enough for the
bacterial growth inside the medium. This method
is used for the quantitative estimation of bacteria,
isolation of bacteria. Study of colony
characteristics and many biochemical
characteristics of bacteria.
19. Sterilized Petridis
Melted agar medium
Bacterial sample
Pipette
Spirit lamp
Incubator
20. Transfer of bacterial sample into Petridis (0.5-1.0
ml)
Pouring of melted agar in the Petridis (12-15 ml)
Mixing the agar with bacterial sample by rotating
the Petridis
Place the agar plate on a level surface
Let the agar to be solidified and then incubate the
plate in an inverted position in
incubator at 350 C for 24-48 hrs
21. Bacterial colonies formed throughout the medium
Colony colour and shape were observed
22.
23. This is a method by which bacteria are cultured on
the surface of solidified agar medium.
This method is used for the quantitative estimation
of bacteria, isolation of bacteria, study of colony
characteristics and many biochemical
characteristics of bacteria
24. Agar plates (previously prepared)
Bacterial sample
Pipette
L-shaped glass rod
Spirit lamp
Incubator
25. Transfer of bacterial sample (0.1 ml) on the agar
surface in the Petridis
Spreading of bacterial sample with the help of L-
shaped glass rod by pushing it back and forth
while rotating the agar plate
Spreading continued until drying of the sample on
the agar surface
Incubation of the agar plate in an inverted position
at 300 C until next class
26. Clear and well separated bacterial colonies formed
on the agar surface
Colony colour and shape were observed
27.
28. The Gram’s staining is the most important staining
procedure used in bacteriology. It is a differential
staining method. It differentiate bacteria into gram
(+) and gram (-). In this procedure bacterial sample
divided into two groups:- first group stain into
purple to violet color while the second group pink
to red. The organism staining violet is called gram
(+) and the organism staining to red is called gram
(-).
31. Solution A: Crystal violet- 20.0g (90% dye
content)
Ethyl alcohol- 20 ml (95%)
Solution B: Ammonium oxalate- 0.85g
Distilled water- 80 ml
Mix solution A and solution B
32. Iodine crystal – 5g
Potassium iodide – 10g
Distilled water- 10 ml
Make the 100 ml stock solution in distilled water
to make the final volume 300 ml.
34. Prepare a smear:- Put a drop of distilled water or
saline on a non greasy clean glass slide.
Aseptically add a little of the colony for staining.
Mix and spread well in the saline or water. Air dry.
Fix by passing the slide 2-3 times through a spirit
lamp flame.
35. Place the slide in crystal violet for 60 secs
Wash in tap water
Place in Lugol’s iodine 60 sec
Treatment with Acetone-Alcohol for 2-3 sec.
Wash in tap water.
Place in safranin for 30 sec.
36. Wash in tap water
Air dry
Observed under oil immersion objective lens on
microscope
37. a. Gram reaction
b. Cell shape
c. Cell arrangement
d. Spore formation
38. Gram-positive anthrax bacteria
(purple rods) in cerebrospinal fluid
sample. If present, a
Gram-negative bacterial species
would appear pink. (The other cells
are white blood cells).
41. The proteobacteria are a major group of Gram-
negative bacteria. Other notable groups of Gram-
negative bacteria include the cyanobacteria,
spirochaetes, green sulfur and green nonsulfur
bacteria.
These also include many medically relevant Gram-
negative cocci, bacilli and many bacteria
associated with nosocomial infections.
42. In the original bacterial phyla, the Gram-positive
forms made up the phylum Firmicutes, a name
now used for the largest group. It includes many
well-known genera such as Bacillus, Listeria,
Staphylococcus, Streptococcus,
Enterococcus,Diplococcus pneumoniae and
Clostridium. It has also been expanded to include
the Mollicutes, bacteria like Mycoplasma that lack
cell walls and so cannot be stained by Gram, but
are derived from such forms.