BACTERIAL INVESTIGATIONS LAB
INVESTIGATION #1: CULTIVATING BACTERIA
MATERIALS:
-
Marking Pen
2 Nutrient Agar Plates
Sterile Cotton Swabs or Toothpicks
Parafilm
Incubator
PROCEDURE:
Day 1: Obtaining the Culture “Streak Plate Method”
Culture Plate #1 (THINK ASEPTIC TECHNIQUE!!)
1. Obtain a sterile nutrient aqar plate. DO NOT open the Petri dish
lid at this time. Turn the plate so the agar side is up. With a
marking pen, label the bottom of the plate with your initials and
date near the edge of the plate (write small). Using a ruler and
marking pen, subdivide the plate into 4 equal quadrants.
2. Choose an ordinary object in the lab or a place on your person to sample. Some suggestions
include: swab your face, tongue (if not sick), under your fingernails, pass it through your hair,
swab the doorknob or your phone.
3. Obtain a sterile inoculating loop. DO NOT touch the loop to anything or lay it down until you are
ready to use it. Collect your culture by rubbing the loop back and forth across the object.
4. Once the culture has been obtained, have your partner lift the agar plate lid high enough to get
the loop under it (both you and your partner need to be holding your breath). Starting at the
top of one marked off quadrant of your plate, “streak” the
inoculating loop back and forth across the agar in a zigzag pattern
ensuring that the loop does not gouge the agar. Leave margins on all
sides of the marked off quarter to limit contamination. Quickly
replace the lid to the plate and place the inoculating loop in the
beakers of disinfectant provided.
5. Choose two other objects to sample and repeat steps 3 and 4.
Leave one quadrant as a control.
6. Label the bottom of your plate with the appropriate sample names
for each quadrant (write small).
7. Obtain a piece of Parafilm and tape your Petri dish closed.
8. Place the Petri dish in the incubator set at 37oC.
Culture Plate #2 (THINK ASEPTIC TECHNIQUE!!)
1. Repeat steps 1-8 from the above directions, but select 3 different bacteria cultures that your
instructor has supplied. You only need to obtain a SMALL sample from the slants.
2. Ensure you label each quadrant with the appropriate letter for each sample.
Day 2: Observation of the Culture
1. Remove the agar plate from the incubator. Observe the plate for isolated colonies of bacteria
and/or mold (“hairy/furry” appearance).
2. Draw your observations for Plate #1 in the first circle on the DATA page and Plate #2 in the
second circle. Record the source of the culture, different colors, shapes, consistencies, and sizes
of the various colonies (mm).
3. Trade plates with another lab group and record observations for their plates.
4. Upon completion, spray a disinfectant (Clorox) into the dish, reseal the dish with Parafilm, and
dispose of the dish as per the instructors directions.
INVESTIGATION #2: EFFECTS OF ANTIBIOTICS
MATERIALS:
- Various Antibiotic Disks
- Bunsen Burner
- Filter Paper Disks
- Forceps
- Inoculating Loop
- Marking Pen
- Ruler (mm)
- Stock Cultures of E. coli and Bacillus megaterium
- Sterile Nutrient Agar Plate
PROCEDURES:
Day 1
Part A: Inoculating the Agar Plate with Bacteria
1. Obtain a stock culture of either E. coli or Bacillus megaterium and a sterile nutrient agar plate.
The instructor will assign your group either E. coli or Bacillus megaterium to test.
2. Turn the agar plate over and lay it on the lab table. Use a marking pen and ruler to draw lines
dividing the plate into 4 equal quadrants.
3. Number the quadrants 1 through 4, placing the numbers near the edge of the dish. Also write
your initials and the letters “EC” or “BM”, depending on whether you use E. coli or Bacillus
megaterium.
4. Obtain a sterile inoculating loop. Do not lay it down on the table or touch it to any object. Place
the inoculating loop into the test tube containing the E. coli or B. megaterium and streak it back
and forth as you are pulling it out of the tube.
5. Open the lid of the sterile nutrient agar plate and inoculate it by
gently spreading the loop over the surface of the agar. Don’t dig into
the agar. Streak from the top to the bottom of the agar, then turn the
plate 90o and steak from top to bottom again. Repeat this step until
you have streaked 3 quadrants, leaving the forth as a control.
Part B: Exposing the Bacteria to Antibiotic Disks
1. Choose an antibiotic to use. Pass forceps through the flame of a Bunsen burner. Using these
forceps, remove 3 antibiotic disks (one at a time) from the dispenser.
2. Evenly space out the 3 disks in quadrant #1 of the nutrient agar plate. With the tip of the
forceps, gently press the disks against the agar until they stick.
3. Record which antibiotic is being used on the plate and the DATA page.
4. Choose a second antibiotic and repeat steps 1-3 for quadrant #2.
5. For quadrant #3, choose a third antibiotic and repeat these steps.
6. Leave quadrant #4 as a control.
Day 2
Part C: Observations of the Inoculated Plate
1. Observe the plate after 24 hours of incubation. White or cloudy areas on the agar indicate
bacterial growth. Notice any clear areas called zones of inhibition surrounding the paper disks.
A clear area indicates that growth was inhibited. Hold the plate at an angle to the light to see
the zones more clearly.
2. Sketch the agar plate on the DATA page.
3. With a millimeter ruler, measure the diameter of the clear zone surrounding each disk. Record
the measurements in the sketch on the DATA page. If no clear zone is present, i.e., the bacteria
are growing right up to the edge of the disc, record the measurement as 0.
4. Trade plates with group members that used a different bacterial culture type and record the
results in the proper place on the DATA page. (Remember to record the types of antibiotics that
the group used.)
INVESTIGATION #3: BACTERIAL MORPHOLOGY AND GRAM STAINING (Identifying Unknowns)
1. Follow the directions on the next few pages to Gram Stain your selected bacteria.
2. You will Gram Stain all 3 bacteria of choice from Investigation #1 Plate #2.
3. Upon successful staining, sketch the bacteria (to scale under oil immersion) in the appropriate
area of the data section. Color the sketch appropriately.
4. Using the handout “Unit 10 Bacteria Investigations Gram Stains of Bacteria A-E” on my website,
correctly identify the stained bacteria (scientific name).
5. Answer the corresponding questions for each bacteria on the data page.
6. Bonus: You will Gram Stain 1 of the 3 bacteria from Investigation #1 Plate #1, identify it, sketch
it, and answer the appropriate questions on the data page.
THE GRAM STAIN
Introduction
Gram's Stain is a widely used method of
staining bacteria as an aid to their
identification. It was originally devised by
Hans Christian Joachim Gram, a Danish
doctor.
Gram's stain differentiates between two
major cell wall types. Bacterial species with
walls containing small amounts of
peptidoglycan and, characteristically,
lipopolysaccharide, are Gram-negative
whereas bacteria with walls containing
relatively large amounts of peptidoglycan and
no lipopolysaccharide are Gram-positive.
It's a mystery
Although it may seem strange, the reason
why bacteria with these two major types of
bacteria cell walls react differently with
Gram's stain appears to be unconnected
with the wall structure itself. The exact
mechanism of the staining reaction is not
fully understood, however, this does not
detract from its usefullness.
The Gram staining method
1. A small sample of a
bacterial culture is removed
from a culture. Using a sterile
inoculating loop, remove a
small colony from your agar
plate. Only a very small
amount of culture is needed; a
visual detection of the culture
on an inoculation loop already
indicates that too much is taken.
2. Place a drop of water onto a
glass slide. The bacterial
suspension is then smeared
onto the glass slide. Spread the
culture with an inoculation loop
to an even thin film over a
circle of 1.5 cm in diameter,
approximately the size of a
dime.
3. Air-dry the culture and fix it
or over a gentle flame, while
moving the slide in a circular
fashion to avoid localized
overheating. The applied heat
helps the cell adhesion on the
glass slide to make possible
the subsequent rinsing of the
smear with water without a
significant loss of the culture. Care must be
taken not to overheat which will char the
cells.
4. Once cool, the slide is
transferred to a support over a
sink and flooded with a stain
called Crystal Violet (a dye
consisting of a methyl
derivative of pararosaniline).
The stain is left on the slide for
about 1 minute. This stains all
the bacteria on the slide a dark purple color.
Note, this stain will not penetrate the waxy
cell walls of some bacteria eg mycobacteria
5. The Crystal Violet is gently
washed off the slide with
running water
6. The bacterial smear is then
treated with Gram Iodine
solution. Add the iodine
solution on the smear, enough
to cover the fixed culture. This
iodine solution reacts with the
Crystal Violet turning it a very
dark shade of blue. It also
causes it to be retained by certain types of
bacteria in a way which is not really
understood.
7. After about 30 seconds the
slide is gently rinsed with
ethyl alcohol which causes
the dye-iodine complex to be
washed out of some bacteria
but not others. This is called
decolorization. Add a few drops
of decolorizer so the solution
trickles down the slide.
If we now looked at the smear down a
microscope, the bacteria which had retained the
Crystal Violet-iodine complex and will appear
blue-black. These are called Gram-positive.
However we would not be able to see those
which had lost the dye-iodine complex which are
called Gram-negative. The final step (#8) in the
Gram stain method is, therefore, to stain the
Gram-negative cells so they can be seen.
8. This is achieved by treating
the smear with a compound
which stains the Gram-negative
cells a color which contrasts
markedly with the blue-black
color of the Gram-positive cells.
The stain common used for this
is either Safrinin or Fuchsin,
both of which are red. These are called
counterstains. Bacteria in the smear which are
Gram-positive are unaffected by the
counterstain.
9. The counter stain is left on
the smear for about 30-60
seconds and then gently rinsed
away with running water.
10. After the counterstain has
been rinsed off, the slide is
placed between some
absorbent paper and the excess
water gently blotted off. Care
must be taken not to rub the
slide with the blotting paper
because this would remove the
adhering bacteria.
11. The slide is gently warmed
to drive off any residual
moisture and then a drop of
immersion oil is placed on
the stained bacterial smear.
This helps transmit light
through the specimen directly
to the high-powered
microscope lens.
12. The slide is then placed on
a microscope stage and the oilimmersion lens (100x) lowered
into the immersion oil. Highpowered lenses are required
because bacteria are very
small.
The Results
Typical Gram-positive bacteria
Gram positive
1.
Staphylococci such as Staphylococcus
epidermidis and Staphylococcus aureus
which is a common cause of boils
2.
Streptococci such as the many species of
oral streptococci, Streptococcus
pyogenes which causes many a sore
throat and scarlet fever and
Streptococcus pneumoniae which causes
lobar pneumonia
3.
Clostridia such as Clostridium tetani
which cause tetanus (lockjaw)
4.
Actinomyces such as Actinomyces
odontolyticus which is found in mouths
5.
Species of the genus Bacillus such as
Bacillus subtilis which are common
microbes living in soil
Staphylococcus epidermidis
Typical Gram-negative bacteria
The bacilli that cause
1. Whooping cough, Bordetella pertussis
Gram negative
2. Typhoid, Salmonella typhi
3. Cholera, Vibrio cholerae
4. The normally benign, ubiquitous, gutdwelling Escherichia coli
Escherichia coli
Generally cocci are Gram-positive but there
are exceptions. The most significant from a
clinical point of view is the gonococcus,
Neisseria gonorrhoea which typically appears
as a Gram-negative diplococcus looking very
much like a pair of kidney bean.
And Finally
SUMMARY
It is important to recognize that not all
species of bacteria can be usefully stained by
Gram's method. Some species are Gram
variable and some what is called Gram
indeterminant. It is also worth mentioning
that the growth conditions may also affect a
bacterial species Gram reaction. For example,
Gram positive cells growing in batch culture
which are into the decline phase of the
growth cycle often show numerous Gram
negative cells present.
1.
Although apparently unconnected with
the bacterial cell wall structure, Gram's
stain differentiates between two major
cell wall types. Bacterial species with
walls containing small amounts of
peptidoglycan and, characteristically,
lipopolysaccharide, are Gram-negative
whereas bacteria with walls containing
relatively large amounts of peptidoglycan
and no lipopolysaccharide are Grampositive.
2.
Examples of Gram-negative bacteria are
Escherichia coli, Salmonella typhi, Vibrio
cholerae and Bordetella pertussis
3.
Examples of Gram-positive bacteria are
Staphylococcus epidermidis,
Streptococcus pyogenes, Actinomyces
odontolyticus and Clostridium tetani
4.
Not all bacteria can be stained by Gram's
method, the best-known exception
belong to the genus Mycobacterium
which have waxy cell walls.
Another major exception is the genus
Mycobacterium which includes such important
human pathogens as Mycobacterium
tuberculosis (TB) and Mycobacterium leprae
(leprosy). Bacteria such as these have very
different cell walls which contain a great deal
of waxy material, although they still contain
peptidoglycan. This waxy material prevents
stain penetration rendering the cells invisible.
Special methods have been devised to stain
these bacteria involving the use heat to allow
the stain to permeate the cell walls. Once
cooled even dilute acid fails to remove it
hence the name "acid-fast" to describe these
species of bacteria.
Since these acid-fast bacteria are not stained
by Gram's method they have been described
as "Gram-negative". Strictly speaking this is
true but it can be misleading to refer to them
this way.
Footnote
The division of all life into prokaryotes and
eukaryotes was challenged by Carl Woese in
1977. He pioneered the use of 16S ribosomal
RNA in the study of taxonomy which was a
major breakthrough in our understanding of
the relationships between all forms of life and
used this technique to reveal major
differences in prokaryotic micro-organisms.
In 1990 Woese proposed that the existing
Kingdom of Prokaryotes be divided into
Archaea and Bacteria. Based on his research,
which showed that these two groups evolved
quite independently from a common
ancestor, the consensus view now is that the
Kingdoms called Prokaryota and Eukaryota
be replaced by three domains (sometimes
called Superkingdoms or Empires) called
Archaea, Bacteria and Eukarya.
STUDENT DATA
INVESTIGATION #2: ANTIBIOTICS AND ZONES OF INHIBITION
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GRAM STAINING
SAMPLE: ____________(Letter)
Scientific Name:
Gram Positive or Negative:
Shape (Latin and English):
Size (include unit):
Nutritional Mode:
Pathogen (If so, to what organisms and with what ailment?):
_____x
SAMPLE: ____________
Scientific Name:
Gram Positive or Negative:
Shape (Latin and English):
Size (include unit):
Nutritional Mode:
Pathogen (If so, to what and what ailment?):
_____x
SAMPLE: ____________
Scientific Name:
Gram Positive or Negative:
Shape (Latin and English):
Size (include unit):
Nutritional Mode:
Pathogen (If so, to what and what ailment?):
_____x
BONUS: ____________ (Object)
Scientific Name:
Gram Positive or Negative:
Shape (Latin and English):
Size (include unit):
Nutritional Mode:
Pathogen (If so, to what and what ailment?):
_____x