Lab 5

MICROBIOLOGY 409

Room 268 SLC

**** Answer questions for Gram staining and Pure Culture****
***** Spore Stain*****
EXERCISE 15
Bacteria Genera Clostridium and Bacillus when in the vegetative stage have a powerful
means of dealing with environmental stress and starvation. Bacillus anthracis, besides others
can undergo a complex developmental cycle that produces endospores covered with a protein
coat exosporium and contain internal calcium dipicolinate that are highly resistant to desiccation
and various insults. The resistant properties of the spores also make them resistant to staining
and therefore staining techniques are harsher and may include heating the sample for a defined
period of time.
Answer all the questions for the spore-staining lab.
Read the procedures carefully before starting.
Modifications:
1. Include a negative control (bacteria that does not produce spores) in your stains
2. Do not perform the final quick spore stain nor the Dorner method; perform only the
Schaeffer-Fulton method
3. Instead of the electric hot plate, use the Bunsen burners and use the smaller ~200 mL
beakers to boil the water.

4. Do not place the beaker directly on the tripod over the Bunsen burner! You need to
place the beaker on the provided square mesh before heating with the Bunsen burner.
5. Use the lab bottle labeled ZNCARBO and not Fushion for the carbolfuchsin stain

Continuing Exercises 14 and 9

****Bacterial staining Gram and Pure Culture****

Both Gram staining and the pure culture techniques are essential tools for the
identification of pathogens. In this laboratory you will practice the gram stain using 4
different bacteria. The technique builds off of the smearing and fixation protocol used in
the previous laboratory. In this lab you will differentiate between gram (pink/red) and
gram + (blue) bacteria. Further you will perform the pure culture, colony isolation
technique. Colony isolation precedes identification of the pathogen.
Exercise 14
Perform the laboratory procedures as outlined except you will not be staining
Mycobacterium smegmatis nor E.coli

On slide 1, make 3 smears on the same slide. On one side of the slide make a smear
with Staphylococcus aureus, on the other side make a Pseudomonas aeregenosa
smear and in the middle mix both cultures
On slide 2, one side Moraxella caterrhalis, other side Bacillus megatarium and middle
mix both cultures
Warning: use ETOH not acid alcohol (H+ ETOH) as decolorizer
Exercise 9
Read the laboratory manual but perform the colony isolation (pure culture) technique as
informed by the instructor (you will not perform the pour method in this laboratory). You
will be given 4 cultures (different bacteria). Each of the different bacteria can be
differentiated from each other solely by analyzing their growth characteristics on
differential media. You will make two mixtures (mix 1 and mix 2, see below for the
bacteria you will mix together)
The pure culture technique allows the separation of different bacteria on a plate.
Basically, you are diluting the sample of bacteria so that you are working with individual
bacteria which then grow into single colonies after overnight incubation. If the growth is
performed on differential or selection plates one can then distinguish the colonies as
growing from a particular bacteria with a specific trait. For example, some differential
media plates will turn the colonies a specific color if the bacteria can ferment a certain
type of sugar. This would allow one to distinguish between bacteria that can or cannot
ferment that particular sugar.
The differential media you will use (Blood, Eosin-methylene blue (EMB) and
MacConkey plates) allow for the differentiation of bacteria that can secrete molecules
that lyse red blood cells or bacteria that can ferment lactose and a host of other
attributes.
Furthermore some plates can be both differential and selective. For example, EMB
plates restrict (the plates are selective) the growth of gram-positive bacteria. In other
words, it allows the growth of gram negative, but not gram-positive bacteria.
Mix 1 - S. pyogenes and S epidermidis (mix these bacteria at a ratio of 1:1 volume)
Mix 2 - Escherichia coli and (Salmonella or Shigella) (mix these bacteria at a ratio of 1:2
volume)
Mix 1 will be streaked for single colonies on NA and blood agar plates.
Mix 2 will be streaked for single colonies on NA, EMB and MacConkey plates.
EXERCISE 9 PURE CULTURE TECHNIQUES

Evaluation of plate and answer questions to hand in.

LAB REPORT 1
HAND IN THE FOLLOWING 2 Documents
Read the introductions to exercises 51-53
1. Fill out Results and answer the questions for exercise 14. Use the loose-leaf
from the lab manual to hand in the results and the answers to the questions (DO
NOT TYPE UP).
2. Answer the questions below after the bacteria-streaked plates have had a
chance to grow: PLEASE TYPE UP and HAND in the answers to these questions
after you have seen the results of the pure culture plating via dropbox (Lab
report 1).
1) Define the term bacterial colony.
2) What is the difference between differential and selective media? Give two
examples of each.
3) Make a Table to describe EMB, MacConkey, Endo, nutrient agar, blood agar,
chocolate agar and HE agar (note that not all these media are for selection or
differentiation).
a. In the top row write MEDIA (for example EMB), KIND of MEDIA (selective,
differential, both, neither, enriched), PURPOSE OF MEDIA (for example,
differentiate between lactose fermenters and nonfermenters), POSITIVE
RESULT (what happens if for example the colonies ferment lactose? Is
there a color change? What color?), DIFFERENTIATION MECHANISM
(how does color change happen?), SELECTION MECHANISM (for
example, what substance in the agar selects for a type of bacteria? What
kills off the bacteria it is selecting against)
4) Describe the colonies seen on each of the plates (For mixture 1 and 2)
MAKE TWO SIMPLE TABLES. One table for MIXTURE 1 AND One table for
MIXTURE 2, WRITING IN THE ANSWERS TO THE FOLLOWING QUESTIONS
WITHIN EACH TABLE.
a. How many different types of colonies can you see growing on the nutrient
agar (NA) plates?
b. How many different types of colonies can you see growing on the EMB
plates?
c. How many different types of colonies can you see growing on the
MacConkey plates?
d. How many different types of colonies can you see growing on the blood
agar plates?

e. In another column write in the colors or appearance of each type of colony

for those colonies.. in other words.. What do you see that makes them
different?.
Example
MIXTURE 1
MEDIA

# of different
colonies

Phenotype of
colonies

NA agar
EMB agar

1
2

white
Green and
white

MacConkey
Blood agar
5) Are any of the colonies on the blood agar plate surrounded by clear areas?
What causes the lysis of the RBCs?
i. Blood agar plate colonies are described in the following manner:
1. Alpha hemolysis: the agar under the colony is dark and
greenish and represents incomplete lysis of red blood cells.
2. Beta hemolysis: red blood cells (RBCs) in the agar are
lysed and you see a clearing around the colonies
3. Gamma hemolysis: There is no lysis of RBCs
6) What are the advantages and disadvantages of the pour plate method as
compared to the streak method for isolating colonies?
7) In the pour method of colony isolation, why must the agar be cooled to 50
degrees Celsius before inoculating it with bacteria?

Lab specialist
EXERCISE 15 SPORE STAIN
1.
2.
3.
4.
5.
6.
7.

Malachite Green
Safranin
Carbolfuchsin (Ziehl's)
Nigrosin
Distilled water in wash bottles
Test tube holders
Bacillus megaterium (24 slants) and another rod-like bacteria that does not produce
spores such as Salmonella or Shigella or E. coli on another 24 Nutrient Agar slants
total of 48
*****Start early4 days for B.m

Media:
???? need more? Nutrient Agar slants - 12 - 23g/L
2.3g + 100ml distilled water
Find a good source of Mycobacterium that acid-fast stains + now for lab 7