1. Fundamental Techniques in Microbiology
Dr Paul D. Brown
BC10M: Introductory Biochemistry

2. Fundamental Techniques
Microscopy
Staining
Aseptic technique
Sterilization and waste disposal
Media preparation

3. Microscopy Measurement Microorganisms are very small Use metric system
Metre (m) : standard unit
Micrometre (m) = 1 x10-6 m
Nanometre (nm) = 1 x10-9 m
Angstrom (Å) = 1 x10-10 m

4. Terms Relevant to Microscopy
For MICR 2909
Lecture 2, 2001
Terms Relevant to Microscopy
Total Magnification
Eyepiece x objective lens
Resolution
Ability of the lens to distinguish two points as separate
Optimal RP achieved with blue light
Theoretical limit for light microscope is 0.2 m
Refractive Index (η)
Measurement of relative velocity at which light passes through a material.
η= 1.0 in air
η (Oil) = η (glass) = up to 1.5
R.P. = 0.5 (lambda) / (η sinØ)
BSc(MolBiol) Lect 2.ppt

5. Resolving Power Optical Instrument Resolving Power R.P. in Angstroms
Human eye
0.2 mm
2,000,000 Å
Light microscope
0.2 m
2000 Å
Scanning electron microscope
5-10 nm Å
Transmission electron microscope
0.5 nm
5 Å

6. Types of Microscopes
Simple: one lens
Compound: more than one lens

7. The Compound Microscope
READ BOTTOM TO TOP!
enters the eye
sees virtual, inverted image
further magnif. by ocular
forms magnified real image
enters objective
focuses light on object
light enters condenser
ocular
objective
object
condenser

8. Objectives 10X Scanning Find the object 40X High-Dry Focus the object
100X Oil immersion Fine focus
(Course focus)
(Fine focus)

9. The Condenser Functions Height of condenser controls
Focus light on object plane
Ensure adequate intensity
Height of condenser controls
Uniformity of brightness
Contrast (minimises “stray light”)
(Indirectly) angle of light entering objective

10. Condenser Height correct too low stray light area  . 1 . intensity
angle
correct too low

11. Use of Immersion Oil R decreased     NA =  sin  = 1,
 > 41 , rays
reflected inside glass (critical angle)
max NA = 0.65
critical angle = 90, wide rays enter objective
max NA = 1.5
= 1,
Air
= 1.5, Immersion oil
R decreased
 
 
NA =  sin 

12. Condenser Iris Diaphragm
aperture
diaphragm
wide aperture
 large , large NA, low R (good resolution), poor contrast (if too wide)
narrow aperture
 small , small NA, high R (poor resolution), good contrast

13. Bright-field Microscope
Contains two lens systems for magnifying specimens
Specimens illuminated directly from above or below
Advantages: convenient, relatively inexpensive, available
Disadvantages: R.P 0.2 m at best; can recognize cells but not fine details
Needs contrast. Easiest way to view cells is to fix and stain.

14. Different magnifications

15. Special Microscopy Applications
For MICR 2909
Lecture 2, 2001
Special Microscopy Applications
Dark Field
Phase Contrast
Fluorescence
Electron Microscope
BSc(MolBiol) Lect 2.ppt

16. Dark Field Microscopy special condenser diaphragm
diffracted light
special condenser diaphragm
occludes direct light, passes wide angle light
angle too wide to enter objective
diffracted light scattered
enters objective
objects light on dark background

17. Phase Contrast Microscopy
For MICR 2909
Lecture 2, 2001
Phase Contrast Microscopy
light rays through objects of different   change in phase, not intensity
special ring-shaped condenser diaphragm
special glass disc in objective
change phase differences to intensity differences
can view transparent
objects as dark on light
background (without staining)
Right; human brain glial
cells
Uses of darkfield and Phase contrast microscopy
View unstained cells
Not subject to shrinkage, artefacts
Some bacteria do not stain easily, e.g., spirochetes (Borrelia, Leptospira, Treponema)
View living cells
Can observe processes, e.g., motility, predation
BSc(MolBiol) Lect 2.ppt

18. Fluorescence Microscopy
For MICR 2909
Lecture 2, 2001
Fluorescence Microscopy
Illuminate specimen with UV  visible fluorescence (filter removes harmful UV)
View auto-fluorescent objects (e.g., chloroplasts)
Stain with specific fluorescent dyes, which absorb in region nm & emit orange, yellow or greenish light
Images appear coloured against a dark background
BSc(MolBiol) Lect 2.ppt

19. Electron Microscopy

21. Stains and Staining Bacteria are slightly negatively charged at pH 7.0
Basic dye stains bacteria
Acidic dye stains background
Simple stain
Aqueous or alcohol solution of single basic dye

22. Simple Stains

23. Differential Stains Gram stain Crystal violet: primary stain
Iodine: mordant
Alcohol or acetone-alcohol: decolourizer
Safranin: counterstain
Gram positive: purple
Gram negative: pink-red
Staphylococcus aureus
Escherichia coli

24. Gram stain – distinguishes Gram+ from Gram -
Gm(+) and Gm(-) both take up CV-I equivalently
CV-I is not readily removed from Gm(+) due to the reduced porosity of the thick cell wall
CV-I is readily removed from Gm(-) thin peptidoglycan due perhaps to the discontinuities in the outer membrane structure introduced during the decolorization step.
-removal of the cell wall (with lysozyme) from a Gm(+) bacterium results in a Gm(-) stain profile

25. For MICR 2909 Lecture 2, 2001 BSc(MolBiol) Lect 2.ppt
Differential Stains
Acid-fast stain
Used to detect Mycobacterium species
Acid fast stain
Acid fast stain (Ziehl-Neelson) for identifying mycobacteria
The lipid mycolic acid (from mycobacteria) is the determinant of retaining the basic fuchsin in the acid-fast stain
BSc(MolBiol) Lect 2.ppt

26. Special Stains
Capsule stain
Klebsiella pneumonia

27. Special Stains
Flagella stain

28. Special Stains
Spore stain (Schaeffer-Fulton)
Bacillus subtilis

29. For MICR 2909 Lecture 2, 2001 BSc(MolBiol) Lect 2.ppt
Aseptic Technique
First requirement for study of microbes
pure cultures, free of other microbes
Maintain a clean environment; work close to the flame
Aseptic Transfers
Purpose Transfer of microbiological cultures from one medium to another sterile medium without contamination of the culture, sterile medium, or the surroundings.
Principle Certain techniques are necessary to handle tubes of media, plated media, and inoculating loops or swabs. Practice in the manipulations, while maintaining aseptic conditions are known as "aseptic techniques." Refer to your laboratory text for acceptable methods of transferring microbial cultures. However, practice is the only way to master the techniques.
Additional Information 1. Gather all the necessary materials (bacterial stock culture, growth media, bunsen burner, transfer tools). 2. Label the tubes properly, following the outlined instructions. 3. Check each organism to be transferred, using the stock cultures. 4. Practice adjusting the flame of the Bunsen burner. 5. Discard contaminated materials properly, referring to the guidelines given in class.
Sterilize the loop or needle by holding in the flame of a Bunsen burner. The metal must glow red before sterilization is considered complete.
When transferring organisms, it is important to maintain a clean environment. Talking, coughing or sneezing should be avoided when performing bacterial transfers from one media to another. Avoid drafts. Maintain a clean environment by disinfecting tabletops before and after working with microorganisms.
Common surface disinfectants are 70% Ethanol and 10% Lysol.
BSc(MolBiol) Lect 2.ppt

30. Streak plate method of isolation
For MICR 2909
Lecture 2, 2001
Streak plate method of isolation
At all costs, there must be prevention of contamination. Often use cotton wool stoppers to flasks, tubes.
Petri dish: ideal for solid medium and allows gaseous diffusion without dust
Development of solid media
needed for colony formation
initially used surface of freshly cut vegetables eg potato
gelatin (1881) used
low melting point (< 37oC)
protein, therefore a nutrient
agar-agar (Hess, Koch’s lab)
polysaccharide - nutritionally inert
melts 100oC ; solidifies ~40oC
Streak Plate Method of Isolation
Purpose The streak plate technique is the most widely used method of obtaining isolated colonies from a mix of cultures.
Principle The streak plate technique is essentially a method to dilute the number of organisms, decreasing the density. This allows for individual colonies to be isolated from other colonies. Each colony is considered "pure," since theoretically, the colony began with an individual cell.
 Additional Information (see also p. 53 in the lab text for diagrams.) 1. Begin with inoculating the first, or primary, quadrant of the agar plate. Use a light touch. Don't penetrate or scrape the agar surface. Cover plate with lid.
2. Flame the loop, cool by touching an uninoculated portion of the surface.
3. Now rotate the plate. Open lid and streak again, following the diagram in the exercise book. Remember: you are picking up growth from quadrant one, and using this as your inoculum for quadrant two.
4. Flame loop; rotate plate, and repeat procedure for quadrants three and four.
The proper wrist action and light touch takes practice.
BSc(MolBiol) Lect 2.ppt

31. Sterilization and Waste disposal
For MICR 2909
Lecture 2, 2001
Sterilization and Waste disposal
Sterilization ensures killing/removal of ALL life forms
Boiling kills most vegetative cells (Bacterial spores unaffected)
Tyndallisation (c.1880): heat, 24hr, heat
Dry heat (very high temperatures)
Moist heat
Autoclave: steam under pressure (121oC)
Filtration (0.45 mm or 0.22 mm filters)
Radiation (Gamma, UV, Ionizing)
Other methods
Sterilization and Waste Disposal
Purpose To sterilize microbiological growth media both before and after inoculation with bacteria.
Principle One method of sterilization is the steam autoclave, which accomplishes the sterilization process by a combination of high temperature, pressure and time.
This is a steam autoclave which operates much like a steam pressure cooker.
Items to be sterilized are placed inside the autoclave chamber.
To sterilize the contents, the autoclave must reach a temperature of 121oC for minutes.
In addition, the pressure must reach 15 lbs. per square inch.
If any of these parameters--time, temperature and pressure--are not met, then sterilization may not be complete.
All microbiological waste, including test tubes, plates, and any other contaminated materials must be autoclaved (sterilized) before disposal.
BSc(MolBiol) Lect 2.ppt

32. Culture media formulation
C & energy source (e.g., glucose)
N source (organic or NH4+ or NO3¯ )
minerals (macronutrients, micronutrients)
Macronutrients
C, H, O, N, P, S - major
K, Ca, Mg, Fe - minor (as cations)
Micronutrients (trace elements)
Mn, Zn, Co, Mo Ni, Cu
(growth factors, vitamins)
(agar)

33. Types of media General purpose Defined
Allows growth of most bacteria, e.g., Nutrient agar
Includes organic C, N, vitamins
May have undefined components e.g., yeast extract, peptone
Defined
All components are pure compounds, not mixtures such as yeast extract
E.g., glucose + (NH4)2SO4 + minerals for E. coli

34. For MICR 2909 Lecture 2, 2001 BSc(MolBiol) Lect 2.ppt Types of media
Selective
Favours one organism and limits growth of others
Lacks some factor(s)
E.g., fixed N, to select for N2-fixing bacteria
Selective toxicity
E.g., bile salts to select for Enterobacteriaceae
Selective via incubation conditions
E.g., gas composition (e.g., N2, 5% CO2, O2), temperature
BSc(MolBiol) Lect 2.ppt

35. Types of media Differential
Different bacteria/groups give different responses
E.g., MacConkey agar: has lactose + peptone + indicator (neutral red)
lactose fermenters  acid  pink colour
non-lactose fermenters use peptone  neutral or alkaline  colourless

36. Enrichment Techniques
For MICR 2909
Lecture 2, 2001
Enrichment Techniques
Increase proportion of desired physiological class
E.g., N2-fixers; cellulose-decomposers; photosynthetic bacteria
Culture mixed population in selective medium and/or conditions
E.g., fixed N-free; cellulose as sole carbon, energy source; anaerobic conditions in light, without organic C
Sample treatment
E.g., boil to kill vegetative cells, leaving spores
BSc(MolBiol) Lect 2.ppt