Fundamental Techniques in Microbiology

Dr Paul D. Brownpaul.brown@uwimona.edu.jm

BC10M: Introductory Biochemistry

Fundamental Techniques

Microscopy

Staining

Aseptic technique

Sterilization and waste disposal

Media preparation

Microscopy

MeasurementMicroorganisms are very smallUse metric systemMetre (m) : standard unitMicrometre (m) = 1 x10-6 mNanometre (nm) = 1 x10-9 mAngstrom (Å) = 1 x10-10 m

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

Resolving PowerOptical Instrument Resolving

PowerR.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 50-100 Å

Transmission electron microscope

0.5 nm 5 Å

Types of Microscopes

Simple: one lensCompound: more than one lens

The Compound MicroscopeREAD 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

Objectives

10X Scanning Find the object

40X High-Dry Focus the object

100X Oil immersion Fine focus

(Course focus)

(Fine focus)

The Condenser

FunctionsFocus light on object planeEnsure adequate intensity

Height of condenser controlsUniformity of brightnessContrast (minimises “stray light”) (Indirectly) angle of light entering objective

Condenser Height

correct too low

stray light

area . 1 . intensity

angle

Use of Immersion Oil

R decreased NA = sin

> 41 , raysreflected inside glass (critical angle)max NA = 0.65

critical angle = 90, wide rays enter objectivemax NA = 1.5

= 1, Air

= 1.5, Immersion oil

NAR

5.0

Condenser Iris 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

aperture

diaphragm

Bright-field MicroscopeContains two lens systems for magnifying specimensSpecimens illuminated directly from above or belowAdvantages: convenient, relatively inexpensive, availableDisadvantages: R.P 0.2 m at best; can recognize cells but not fine detailsNeeds contrast. Easiest way to view cells is to fix and stain.

Different magnifications

Special Microscopy Applications

Dark Field

Phase Contrast

Fluorescence

Electron Microscope

Dark Field Microscopy

special condenser diaphragmoccludes direct light,

passes wide angle lightangle too wide to enter

objective

diffracted light

diffracted light scatteredenters objectiveobjects light on dark background

Phase Contrast Microscopylight rays through objects of different change in phase, not intensity special ring-shaped condenser diaphragmspecial glass disc in objective change phase differences to intensity differences can view transparent

objects as dark on lightbackground (without staining)

Right; human brain glialcells

Fluorescence MicroscopyIlluminate 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 230-350 nm & emit orange, yellow or greenish light

Images appear coloured against a dark background

Electron Microscopy

Stains and Staining

Bacteria are slightly negatively charged at pH 7.0Basic dye stains bacteria Acidic dye stains background

Simple stainAqueous or alcohol solution of single basic dye

Simple Stains

Differential Stains

Gram stainCrystal violet: primary stain Iodine: mordantAlcohol or acetone-alcohol:

decolourizerSafranin: counterstainGram positive: purpleGram negative: pink-red

Staphylococcus aureusStaphylococcus aureus

Escherichia coliEscherichia coli

Gram stain – distinguishes Gram+ from Gram -Gram stain – distinguishes Gram+ from Gram -

Gm(+) and Gm(-) both take up CV-I equivalentlyGm(+) and Gm(-) both take up CV-I equivalently

CV-I is not readily removed from Gm(+) due to the reduced CV-I is not readily removed from Gm(+) due to the reduced porosity of the thick cell wallporosity of the thick cell wall

CV-I is readily removed from Gm(-) thin peptidoglycan due CV-I is readily removed from Gm(-) thin peptidoglycan due perhaps to the discontinuities in the outer membrane perhaps to the discontinuities in the outer membrane structure introduced during the decolorization step.structure introduced during the decolorization step.

-removal of the cell wall (with lysozyme) from a Gm(+) -removal of the cell wall (with lysozyme) from a Gm(+) bacterium results in a Gm(-) stain profilebacterium results in a Gm(-) stain profile

Differential Stains

Acid-fast stainUsed to detect Mycobacterium species

Special Stains

Capsule stain Klebsiella pneumonia

Special Stains

Flagella stain

Special Stains

Spore stain (Schaeffer-Fulton)

Bacillus subtilis

Aseptic Technique

First requirement for study of microbespure cultures, free of other microbes

Maintain a clean environment; work close to the flame

Streak plate method of isolation

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 m or 0.22 m filters)Radiation (Gamma, UV, Ionizing)Other methods

Culture media formulationC & energy source (e.g., glucose)

N source (organic or NH4+ or NO3¯ )

minerals (macronutrients, micronutrients)Macronutrients

C, H, O, N, P, S - majorK, Ca, Mg, Fe - minor (as cations)

Micronutrients (trace elements)Mn, Zn, Co, Mo Ni, Cu

(growth factors, vitamins)(agar)

Types of media

General purpose 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

Types of mediaSelectiveFavours one organism and limits growth of othersLacks some factor(s)

E.g., fixed N, to select for N2-fixing bacteria

Selective toxicityE.g., bile salts to select for Enterobacteriaceae

Selective via incubation conditionsE.g., gas composition (e.g., N2, 5% CO2, O2),

temperature

Types of media

DifferentialDifferent bacteria/groups give different responsesE.g., MacConkey agar: has lactose + peptone +

indicator (neutral red) lactose fermenters acid pink colournon-lactose fermenters use peptone neutral or

alkaline colourless

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