Transcript Pure Culture - IRSC Biology Department

**Turn on Incinerators and hot plates (low heat) **
Pure Culture Techniques

Method of multiplying microbial organisms by
letting them reproduce in a predetermined
culture media under controlled laboratory
conditions

Agar: jelly like substance derived from
seaweed; thickening agent
 We use agar because most microorganisms cannot digest
agar so it provides a firm surface on which to grow and we
can pick out individual colonies

Broth: liquid media (same as agar without the
thickening agent)

We can add specific ingredients to agar to grow
or inhibit specific microorganisms




Selective: inhibit or help growth of certain
organisms with the use of specific chemicals
Differential: organisms produce characteristic
changes or growth patterns dependent on the
ingredients present
Supportive: supports the growth of most organisms
Enrichment: supplemented with highly nutritious
materials that allow for the growth of fastidious
(picky) organisms

Tryptic Soy Agar (TSA) : Supportive media;
general media used to grow most
microorgansims

Eosin Methylene Blue (EMB): Selective and Differential




Selects for Gram negative organisms (inhibits the growth of Gram
Positive organisms)
Has Eosin Y and Methylene Blue- indicator dyes that react
with any acidic products resulting from lactose fermentation
to color the colonies
Lactose fermentation causes precipitation of the dyes on the
surface of the colonies resulting in different colorsDifferential



Large amounts of acid → green metallic sheen
Small amounts of acid → pink
No fermentation → colorless


Needed to identify bacteria and for antibiotic
sensitivity
Can be achieved by:
Pour plates
 Isolation Streaks


Pour Plate: Serial dilution of the original sample is
performed and a small amount of the final dilution is
added to melted agar. The melted agar is poured into
an empty sterile plate. Colonies will develop
subsurface.


Streak Plate: the original culture is directly diluted
across an agar surface using an inoculating loop
The main idea of a pure culture is to dilute or thin out the
original sample until the organism of interest is isolated
and pure.


Split up into groups of 2
1 TSA plate/group


1 EMB plate/group


Mix 1 (Room Temp)
Mix 2 (37°)
LABEL YOUR PLATES
Name/Initials
 Class Section
 Which mix you used

Exercise 6


The way a smear is prepared will determine
how well your stain will come out
3 goals to a good smear
Making sure the cells adhere to the slide- heat fix
 Insure that shrinkage of the cells does NOT occurdistorts the cells, give improper representation of the
cells shape/size-done by air-drying your slide before
heat-fix
 Prepare a thin smear- thick smears make it harder to
see individual cells and their arrangement, gives
false staining results


Solid media
Add one drop of water to your slide
Use sterile inoculating needle to pick one colony- just a slight
touch needed
 Mix bacteria into water onto slide and try to spread out and thin as
possible



Liquid media: no additional liquid is needed
Use a sterile loop, place a couple loopfuls of broth onto the slidesterilize loop between touching the slide and dipping your loop
 Place slide on heat plate to dry and heat fix smear (just until slide
is completely dry)


Make sure you label your slide so you know which smear is
which organism

Different types of Staining




Simple Stain: use of a single stain to color a bacterial cell
Differential Stain: Use of a combinations of stains to
differentiate between 2 or more organisms
Structural Stains: stains only one part of a cell so it can be
distinguished from the rest of the cell (ex: flagellar)
How do the stains work?




Biological Stains contain chromophores- chemicals that can
impart color
A bacterial cell has slight over-all negative charge
Cationic/Basic Dyes: positively charged so it binds to the cell
Anionic/Acidic Dyes: negatively charged; repels the cells,
stains everything else, results in a negative/indirect stain


Uses only one color
Can only determine size and shape

Uses more than one color with a decolorization
step between them. (will do next lab)

Pure Culture-Split up into groups of 2
TSA (1/Group)Mix 1 (Incubates at Room Temp)
 EMB (1/group)  Mix 2 (Incubates at 37º)
 Isolation Streak of the assigned mix on each plate


Smear Prep & Simple Staining- On your own
Pseudomonas aeruginosa in TSB
 Staphylococcus aureus on TSA
 Stains:





Carbolfuchsin: 1 min
Crystal Violet: 1 min
Safranin: 15 min
Methylene Blue: 15 min

Work on your own. Each student to make:



Each of student in your row will use a different stain






Smear of Escherichia coli from TSB (tryptic soy broth)
Smear of Staphylococcus aureus from TSA (tryptic soy agar)
Carbolfuchsin: 1 min
Crystal Violet: 1 min
Safranin: 15 mins
Methylene Blue: 15 mins
Make notes of your observations; make sure you take a
look at your fellow classmates slides to see the different
stains
Record your observations on page 43