Caitlin Wheeler Microbiology Skills Notes 2018

Microbiology 214

Contents:

• General Theory
• Aseptic technique
• Microscope
• Bacterial smears, staining and heat fixing
• Simple staining
• Wet mount
• Endospore staining
• Gram staining
• Capsule negative staining
• Flagella staining
• Acid fast staining
• Growth Media
• Preparing Media
• Pouring Agar plates
• Spread plates
• Streak plates
• Autoclave
• Pipetting
• Dilution series
• Pour plate
• Counting Microbial cells

BLUE HIGHLIGHT = Theory

GREEN HIGHLIGHT = HOW TOs and Practical components

Caitlin Wheeler Microbiology Skills Notes 2018

GENERAL THEORY and OTHER NOTES

NOTE: SKILLS TEST WE WILL BE GIVE INOCULATION, SPREAD PLATE AND

STREAK PLATE

- Invert petri dishes in order to avoid condensation

• Labelling of Petri Dishes
INITIALS
DATE
TYPE OF PLATE and ABBREVIATION OF CULTURE (e.g. E. coli = “EC”)

Q: Will inoculating a test tube with liquid media with 0.1 mL of a bacterial
suspension using a micropipette and inoculating a different test tube with
liquid media with one inoculating loopful of the same bacterial suspension
yield the same results? Explain
Yes, the same level of turbidity will be observed in both test tubes with
no significant difference between the two.
The same amount of growth is observed even though different initial
volumes of the bacterial suspension were inoculated into the media due
to the microorganisms utilising the nutrients available to them and will
not stop replicating until the growth media is completely used up.
Therefore, the test tube which had 0.1mL inoculated into it will reach
the stage in less time than the test tube which had one loopful
inoculated into it but ultimately, they will both reach the same level of
turbidity.

• Shapes of Bacteria
Spherical Coccus
Rod-shaped Bacillus
Spiral Spirillum
Comma Vibrio
Irregular Pleomorphic
Caitlin Wheeler Microbiology Skills Notes 2018

micrococcus

• Arrangements of Bacteria
Pair Diplococcus
Chain Streptococcus
Cluster Staphylococcus
Square Micrococcus
Cube Sarcina
Chain Streptobacillus

• Colony characteristics
Caitlin Wheeler Microbiology Skills Notes 2018

• Aseptic Technique:
Work close to Bunsen burner when working with container, bottle, test
tube or petri dish
Glass test tube or bottle should be run through the flames after opening
and before closing it
Sterilize equipment by flaming it in Bunsen burner or placing in 70%
ethanol
REMEMBER TO VORTEX ALL TEST TUBES WITH CULTURES IN THEM

HOW TO:
• Inoculation loop: Liquid media
1. Light Bunsen burner
2. Vortex test tube
3. Take loop and at a 45’ angle flame in Bunsen burner until glows
red
4. Remove cap of test tube using pinky finger
5. Flame top of test tube
6. Insert loop and cool off on side of glass
7. Insert into media and “mix” around
8. Perform culture streak

• Inoculating loop: Solid media

1. Light Bunsen burner
2. Take loop and at a 45’ angle flame in Bunsen burner until glows
red
3. Open petri dish slightly
4. Insert loop and cool off on side of glass
5. Select ONE PURE COLONY
6. Insert loop into colony
7. Perform culture streak

Q (4): What is the difference between a spread plate and a pour plate
A spread plate places cells directly onto the surface of the nutrient
source (agar) which after incubation allows for further analysis of cells
(grown of surface)
A spread plate has a change in its dilution factor
0.1 ml
A pour plate places cells into the subsurface of a nutrient source (agar)
which allows CFUs to form but doesn’t allow for further analysis
1 ml
Caitlin Wheeler Microbiology Skills Notes 2018

A pour plate has no change in its dilution factor

• Culture media
Growth, transport and storage (Liquid or solid)
Nutrients requirements
- Synthetic or Defined media = precise composition of all components
is known
- Complex or Undefined media = components of unknown composition
AGAR- melts when autoclaved and solidifies at 48 oC
a sulphide polymer consisting mainly of D-galactose, 3,6- anhydro-
L-galactose and D-glucuronic acid
red seaweed

• Types of media
- Selective Medium: promotes the growth of particular micro-
organisms
- Differential Medium: distinguish between bacterial groups. Initial
identification of microorganisms based on biological character
properties
- Enrichment Medium: used to isolate species of interest in the lab
Caitlin Wheeler Microbiology Skills Notes 2018

Practical 1

• Labelling and functions of microscope parts

Ocular lens: Contains the ocular lens 10x to 15x magnification

Arm: Supports microscope, use when being carrier
Nonius Scale: Ruler which will only be used for larger organisms in
Zoology not microbes
Stage: Flat platform which supports the slide being analyzed
Large Adjustment Knob: Coarse focus, moves stage up or down
Fine Adjustment Knob: Fine focus, moves stage up or down in small
increments
Base: Supports microscope and is used when carrying microscope
Objective lens :4x 40x or 100x magnification power [eyepiece ocular
x objective = magnification]
Slide holder: holds slide in place for viewing (stage clips)
Iris Diaphragm: Controls intensity and size of light cone projected
Condenser: Aids in focusing the light onto the sample being analyzed
Light source: Projects light upwards through Iris Diaphragm, Lenses and
Slide

OIL USED FOR 100x OBJECTIVE LENS (Oil immersion Objective)

• Microscope
(See page 18 in practical manual for detailed explanation on how to use
microscope)
NB points:
• use plastic ring to turn objective lenses into and out of position over
slide not the sliver metal lenses
Caitlin Wheeler Microbiology Skills Notes 2018

• IN SKILLS TEST DO NOT ADJUST IRIS DIAPHARGM OR CONDENSER

Calculating magnification:
• Multiply the ocular and objective lenses to give total magnification
• Total Magnification=ocular x objective
Microscope Resolution:
• d = 0,5 ʎ / nsinƟ
• Where d is the microscope resolution, 0,5 is a constant, ʎ is
wavelength which will be given, and nsinƟ is the numerical aperture
value read off the objective lens [ _x/ nsinƟ value ]

Q: What is the microscope resolution of a 10x, 40x and 100x objective is the
wavelength is 530 and their respective numerical openings/apertures are 0.25,
0.65 and 1.25
A: 10x 1060/40x 407,69/100x 212

• Sample Preparations- Bacterial Smears for Staining and Heat Fixation

Fixed and stained to increase visibility of cell structures
NOT USED for Capsule or Flagella study as heat fixing causes
morphological changes

HOW TO:
1. Vortex test tube, sterilize loop
2. Place droplet of culture onto slide and “mix” around or smear
3. Wait to dry
4. Heat fix by running through Bunsen burner three times
5. Stain

Q: How would one fixate Prokaryotes versus Eukaryotes in order to preserve

their shape?
• Prokaryotes are fixated using heat
• Eukaryotes are fixated using chemicals in order to preserve their
lager delicate structures

• Simple Staining
Use of a single dye, increases visibility of cell structures bu increasing the
contrast between cells and background
Caitlin Wheeler Microbiology Skills Notes 2018

HOW TO:
1. Heat fix a bacterial smear
2. Cover smear with BASIC CRYSTAL VIOLET and let it stand for 20-
30s
3. Rinse off slide using water
4. Examine slide > oil immersion

Q: What are the primary features of a dye? (2)

• Chromophore groups which give the color due to their conjugated
double bonds
• Can bond to cells via ionic, covalent or hydrophobic bonding

• Preparation of Wet Mount

Used to observe MOTILITY (as organism ages so motility decreases)

HOW TO:
1. Clean slide if dirty with ethanol
2. Transfer a drop of suspension solution onto slide aseptically (Do
not use saline solution as suggested by the Prac Book)
3. Place edge of coverslip onto slide (touching liquid)
4. Lower coverslip NO BUBBLES
Caitlin Wheeler Microbiology Skills Notes 2018

Practical 2

• Endospore Staining
Differential stain (each of the dyes used stain the cell components a
different color)
Schaeffer-Fulton method involves heat aiding in pushing the malachite
green stain into the endospore, then counter staining using light red
safranin
Endospore = green
Other cell components = light red

HOW TO:
1. Transfer culture onto slide aseptically (Can be liquid or solid
medium)
2. Spread out mixture
3. Let air dry- might take a while but don’t rush
4. Heat fix by running slide through flame +- 3 times
5. Perform endospore stain:
6. Place blotting paper on slide over water bath
7. Saturate slide with malachite green stain
8. Steam for 5 minutes
9. Remove blotting paper and remove from over water bath
10. Rinse with water for 30s to cool
11. Counterstain with safranin for 1 minute
12. Rinse with water for 30s
13. Gently blot with paper towel to remove excess liquid

Q: What are the different positions of an endospore?

• Swollen Sporangium
• Central
• Subterminal
• Terminal

• Gram Staining
Differential stain
If not clear then can use KOH test (not used often but effective)- mucoid
string produced with Gram negative bacteria when a loop is pulled
through the suspension
Caitlin Wheeler Microbiology Skills Notes 2018

HOW TO:
1. Transfer culture onto slide aseptically (Can be liquid or solid
medium)
2. Spread out mixture
3. Let air dry- might take a while but don’t rush
4. Heat fix by running slide through flame +- 3 times
5. Perform Gram stain:
6. Place crystal violet on top of dried mixture for 20s
7. Wash with water for 2s
8. Place Gram’s iodine on top for 1 minute
9. Decolorize with alcohol for 10-20s (flow colorless)
10. Wash with water for 2s
11. Stain with safranin for 20s
12. Wash with water for 2s
13. Gently blot with paper towel to remove excess liquid
14. Observe slides under microscope
15. PINK/RED = Gram NEGATIVE
16. PURPLE = Gram POSITIVE = Peptidoglycan

Q: What is the mechanism of Gram staining? (10)

o Stain cells purple using crystal violet for one minute. Water
rinse
o Stain with iodine (mordant) for 1 minute. Water rinse. Cells
remain purple
o Decolorize cells using alcohol for 10-30 seconds. Water
rinse (Gram- positive= purple, Gram-negative= colourless)
o Stain with Safranin which is the counterstain for 30-60
seconds. Water rinse. (Gram-positive= purple/ Gram-
negative= red)
Caitlin Wheeler Microbiology Skills Notes 2018

• Capsule Negative staining

No heat fixing required due to shrinking of cell
Nigrosine or India ink
Unstained area = capsule (could also be result of separation of cell and
ink during drying therefore not entirely accurate)

HOW TO:
1. Transfer 2-3 drops cell suspension onto slide aseptically
2. Add small drop of nigrosine solution and mix into culture with
loop
3. Hold a different slide at an angle of 45’ and drag from one end of
the slide with the culture on it to the other side (Thin smear)
4. Allow slide to air dry fully
5. Study smear under microscope using 100x objective and oil
immersion

• Flagella Staining
[Was a demonstration in class therefore WONT to be asked to perform-
theory and identifying more important]
Unstained only visible under electron microscope
Motility in wet mount= flagella
Number and arrangement > need to be stained
Mordants increase diameter (tannic acid and potassium alum)
Basic stain (fuschin or silver nitrate)

Arrangement
Caitlin Wheeler Microbiology Skills Notes 2018

• Monotrichous
• Iophotrichous
• Amphitrichous
• Peritrichous

• Acid Fast staining

Differential stain
[Was a demonstration in class therefore WONT to be asked to perform-
theory and identifying more important]
Mycobacterium sp. (leprosy and tuberculosis)
Mycolic acids prevent normal dyes from binding due to branched
hydroxyl fatty acids in cell wall
*Ziehl-Neelsen method: Heat and phenol used to drive in basic fuschin
RED
NOT easily decolorized (even when using acid alcohol)
Counterstain surrounding cells with second dye
Caitlin Wheeler Microbiology Skills Notes 2018

Practical 3

• Growth media
Types:
Liquid (no agar) = Turbidity (opaqueness/cloudy), Pellicle formation
(mass on-top of liquid), Sediment formation (mass at bottom),
Slime production indicate GROWTH
Semi solid = motility and oxygen requirements
Solid (High agar %) = CFU (colony forming units), colony
characteristics, pure
cultures obtained and stored, biochemical testing

Q: What are the factors that need to be considered for the growth of
microorganisms/ Growth requirements
Ideal temperature, nutrient supply sufficient, correct pH, correct osmotic
pressure

Q: What is the difference between synthetic and complex medium

Synthetic or defined media is when the exact chemical components of
the media are known
Complex media is when the exact chemical components of the media
are unknown

• Making up/preparing media

[Was a demonstration in class therefore WONT to be asked to perform-
theory and identifying more important]
Indicated on bottles:
Broth powder= 16g/ 1L media (1.6g/100mL media)
Bacteriological agar= 12g/ 1L media (1.2g/100mL media)
Add required mass into flask, add 100mL volume using distilled water,
seal with cotton wool plug and cover with tin foil, place on trolley for
autoclaving

• Pouring agar plates

HOW TO:
1. Collect autoclaved nutrient agar from 50oC water bath
2. Remove lid and flame top
3. Pour agar into BOTTOM (smaller plate) of petri dish near Bunsen
burner
4. Once set invert the plates and incubate at 37oC
Caitlin Wheeler Microbiology Skills Notes 2018

Q: How would one avoid contamination while pouring agar plates?

Avoid contamination by working close to the flame (zone of sterility),
only opening the petri dish a small amount, working aseptically

• Spread plate
Isolate single bacterial colonies from cell suspension CFUs (Colony
forming units)

HOW TO:
1. Label agar plate [initials, spread plate, date, abb. of microorganism]
2. Pipette (micro or normal) 0.1ml (1000 µm)
3. Release pipette onto MIDDLE of agar plate
4. Flame ethanol sterilized glass hockey stick and wait until flame stops
5. Open petri a small amount near the Bunsen burner
6. Cool down hockey stick on top half of petri dish
7. Use hockey stick to move liquid culture around making sure no gaps
are present
(Use thumb to turn petri dish around in a circular pattern while
pressing down slightly on agar with hockey stick)
8. Invert plate once dried and incubate at 37 oC

• Streak plate
Inoculation loop, creating dilution of cells between quadrants
Final quadrant should show individual CFUs
Once inoculum is used don’t return and use it again- “dilute culture”
Loop- use the edge of loop as shown in the picture on the right

HOW TO:
1. Label agar plate [initials, streak plate, date, abb. of microorganism]
2. Create 4 quadrants using marker as follows in the picture
3. Vortex test tube if liquid media is being used
4. Heat inoculation loop in flame until red hot (at 45’ angle)
5. Remove test tube cap with pinky finger
6. Flame top of test tube
7. Place loop inside test tube and cool on side
8. Mix around within the media
9. Remove loop and flame test tube top again, replacing cap
10. Bring agar plate into zone of sterility and open lip slightly
11. Draw 3 straight lines on agar from 1 to 2 side to side
12. Flame inoculating loop
Caitlin Wheeler Microbiology Skills Notes 2018

13. Cool loop in center of agar

14. Draw 3 lines from quadrant 2 to 3
15. Flame loop
16. Cool loop in center of agar
17. Draw 3 lines from quadrant 3 to 4
18. Flame loop
19. Draw a “squiggle” from quadrant 4 into center/quadrant 1 (this will
hopefully give single CFU)
20. Flame loop and replace
21. Invert plate and incubate at 37oC

• Autoclave
121oC
15 Pound force per Square inch (PSI)
Used to sterilize (no living organisms present) using heat, steam and
pressure
Autoclave tape- turns black when exposed to high temps
• Slow exhaust = solutions/liquids and medical waste - Agar
(water doesn’t boil over into flask)
• Fast exhaust = empty glassware and instruments
Caitlin Wheeler Microbiology Skills Notes 2018

Practical 4

• Pipetting
< 1mL
Microliters 1000yl = 1 ml
P20 (0.5-20), P200 (20-200) – use yellow sterile plastic tips
P1000 (200-1000) – use blue sterile tips

HOW TO:
1. Do not exceed limits of micropipettes
2. Set meter to desired volume CONVERSION of 1 ml is equal to 1000 yl
3. Attach disposable tip by inserting the shaft directly into it from the
sterile box
4. Push down the plunger down to the first “stop”
5. Insert the tip into the liquid
6. Release plunger slowly – draws up liquid
7. Insert micropipette into test tube
8. Push down to second “stop” – expels all liquid
9. Remove tip by pressing tip ejector into yellow bucket
10. REPLACE TIP BETWEEN EACH PIPETTE- avoid contamination

• Dilution series

HOW TO:
1. Label test tubes with dilutions (10-1, 10-2, etc.) containing sterile
saline solution
2. Using a micropipette transfer 1ml (1000yl) of broth mixed culture
into first test tube (10-1)
3. Vortex
4. Change tip
5. Transfer 1ml from 10-1 first test tube and transfer into second test
tube (10-2)
6. Vortex

• Pour plate
Dilution series
Add set volumes of each dilution to separate petri dishes
If pure cultures are needed – streak plate following pour plate
Caitlin Wheeler Microbiology Skills Notes 2018

HOW TO:
1. Label petri dishes
2. Start with more diluted test tube
3. Micropipette 1ml (1000yl) of 10-2 test tube into middle of petri dish
4. DO NOT CHANGE TIP
5. Micropipette 1ml (1000yl) of 10-1 test tube into middle of a different
petri dish
6. Take melted agar from 50 oC water bath
7. Pour agar into petri dishes aseptically (near flame and don’t open
petri dish widely)
8. CAREFULLY swirl petri dishes in a figure of 8 pattern = do not spill on
lid
9. Wait for agar to set and then invert and incubate at 37 oC
Caitlin Wheeler Microbiology Skills Notes 2018

Practical 5
Direct Microscopic Methods: both alive and dead cells counted

Stained Smears Set volume of a bacterial suspension is smeared onto a

set area on a slide. The slide is fixed (heat or chemical)
and stained
Repeated using different areas. Average of results is
calculated as the microbes per ml
Special Counting Gives info on size and morphology. Inexpensive and fast
Chambers Grid etched into slide, any dilution can be used,
chambers volume
Petroff-Hausser: 0.2 mm bacteria (small)
Hemocytometers: 0.1 mm larger eukaryotic
microorganisms e.g. Yeast
Culture Methods: survive experimental process of incubation or culturing in
a medium are counted

Dilution Plate TFTC (too few to count) 25-250 TNTC (too numerous to
Counting method count)
Inaccuracy: not enough dispersion, medium can’t
support all viable cells in culture, hot agar kills cells
Pour plate: dilution factor not changed from test tube to
petri dish
Spread plate: dilution factor changed
Cylindrical tube Smaller volumes compared to dilution plate method.
method Thin layer of agar on inner surface of tube- the colonies
grow on this layer and can be counted using special
apparatus. Anaerobic bacteria counting
Membrane filter Demonstration: Used for counting low numbers of
method bacteria in WATER SAMPLES. Filter using sterile
membrane with pores of 22 µm (retains bacteria).
Specialized agar plate- diffusion of nutrients through
membrane. Petri dish is not inverted when incubated
Unit: CFUs per ml of water filtered
Air Samples Equipment used to draw in certain volume of air into
petri dishes. Viable cells drop and grow on agar forming
CFU
Unit: Number of cells per cubic meter of air
Physical and Chemical Methods: counting microbes in a culture, large
number
Caitlin Wheeler Microbiology Skills Notes 2018

Determination of Centrifugation of medium gives dry mass of cell material

dry mass of cell which is then resuspended in a buffer. Specific volume of
material the final suspension is dried in oven 105oC (until reaching
constant mass). Buffers salt contribution should be
accounted for in calculations
Determination of Centrifugation of medium. Dry mass suspended in buffer
nitrogen content (NOT Nitrogen containing). Nitrogen content is
of cell material determined (approx. 14% of dry mass)
Turbidity Both dead and alive cells are counted- disadvantage.
determination 10 mil cells per ml = turbidity. Absorbance of light
increases and the turbidity increases and so less light
passes through. Spectrophotometer
Determination of Indirect method but effective- cell concentration is equal
substrate or to the conversion of substrate to products
product
conversion
through
metabolism

• Counting Chambers
Average number of blocks=
Petroff-Hauser:
Used to count bacteria
0.02 mm depth
Number of cells in 1 ml = average number of blocks x 2 x 107 cells/ml

Hemocytometer:
Used to count yeast
0.1 mm depth
Number of cells in 1 ml = average number of blocks x 4 x 106 cells/ml

• Dilution Plate
25-250 colonies
Below 25= TFTC (too few to count)
Above 250= TNTC (too numerous to count)
Petri dishes used for pour plates
Nutrient agar plates used for spread plates
Caitlin Wheeler Microbiology Skills Notes 2018

• Turbidity determination
Spectrophotometer
Absorbance is directly proportional to cell concentration up to 1
Advantage= if a standard curve has been drawn for the organism then
quicker than plate-counting
Different standard curves for different organisms
(same optical density= different number of cells per ml)
Density of cells needs to be above 107 cells per ml

HOW TO:
1. Serial dilution
2. Label test tubes: 1 , 1/2 , 1/4 , 1/8 and 1/16
3. Transfer 3 ml of bacterial suspension provided into test tube 1
4. Transfer 3 ml of Nutrient sterile broth into the 4 other test tubes
5. Transfer 3 ml of bacterial suspension into ½ test tube (dilution 1:2)
6. Discard pipette tip
7. Transfer 3 ml from test tube ½ into test tube ¼ (dilution 1:4)
8. Discard tip
9. Repeat in sequence from test tube ¼ into test tube 1/8 and from 1/8
to test tube 1/16 remembering to discard tip
10. Spectrophotometer
11. Turn on
12. Set wavelength to 550 nm (ʎ)
13. Pick up cuvette holding blank on the FROSTED SECTION
14. Place cuvette holing the BLANK into the holder ARROW FACING
BACK
15. Set absorbance to 0
16. Mix dilutions before next step by swirling/vortexing
17. Add dilutions into the empty cuvette one by one (1, 1/2, 1/4, 1/8,
then 1/16)
18. Measure absorbance value by placing cuvette into
spectrophotometer individually
19. Wait for absorbance value to stabilize
20. Record absorbance value (value will decrease from 1 (undiluted) to
1/16)

• Membrane Filtration
Microbial cell counts in large volumes of liquid
Expensive and sometimes contamination is too high (overgrowth on
membrane)
Caitlin Wheeler Microbiology Skills Notes 2018

E. coli = green
Coliforms = yellow
Demonstration of 100 ml rainwater passing through 0.45 µm filter. Filter
placed on a MLGA medium plate