Analytical Microbiology

- analytical instrumentation in microbiological research and applications which include

Gas chromatography FA analysis/Quinolone Analysis GC- mass spectrometry PCR HPLC/Ion chromatography TLC RAPD Miscroscopy Electrophoresis

- analysis of microbial fermentation products, biotransformations, biodegradation of wastes or heavy metals

- analysis of chemical markers used in the identification and taxonomy of microorganisms

Familiarization of different analytical software and methods of data analysis and interpretation

Anton van Leeuwenhoek (1673)

Animalcule

- using a simple microscope, he was the firstto observe microorganisms.

Leeuwenhoek’s drawings of bacteria

A, C, F and G are rod –shapedE – spherical or coccus-shaped, H cocci packets

Robert Hooke (1665) - observed that plantmaterial was composed of little boxes, heintroduced the term “ cell.”

Cell walls in cork tissue

Hooke’s observation laid the groundwork for developmentof the cell theory, the concept that all living things arecomposed of cells.

Born on July 18, 1635

Drawing of Robert Hooke, which represents one of the first microscopic descriptions of microorganismsA blue mold growing on the surface of leather, the round structures contain spores of the mold

Microscope = 2 Greek words mikros = small skopein = to look through

Three lenses/light

1. Ocular/eyepiece 2. Objectives 3. Condenser

LPO = 10x (100)HPO = 40/45x (450)OIO = 100x (1000)Scanner = 4x (optional)

Together, the optical and mechanical components of the microscope,including the mounted specimen on a glass micro slide and coverslip, form an optical train with a central axis that traverses the microscope base and stand

Microscope must accomplish 3 tasks:

1. produce a magnified image of the specimen 2. separate the details in the image 3. and render the details visible to the human eye or camera

Coxial binocular microscope

Dissecting microscope

Inverted microscope

Inclined microscope

Microscope with cleaning kit

Projection microscope

Research projection microscope

Zoom tinocular microscope

Compound microscope

Resolving Power

- measures the ability to distinguish small objects close together 0.61 (lambda) r.p. = ______________ (N sinØ)

where lambda = wavelength of illuminating light

for light scope, can improve R.P. by making lambda smaller or sinØ larger

R.P. is smallest for violet light, human eye is more sensitive to blue, optimal R.P. is achieved with blue light (450 nm).

n sinΦ is called numerical aperture (it measures how much light cone spreads out between condenser and specimen).

more spread = better resolution Φ = angle of light cone maximum value is 1.0

n = refractive index n = 1.0 in air can increase with certain oils (up to 1.4), called immersion oil N.A. is property of lens

Theoretical limit of R.P. for light scope is 0.2 micrometers

Optical Instrument Resolving Power RP in

Angstroms

Human eye0.2 millimeters (mm)

2,000,000 A

Light microscope0.20 micrometers (µm)

2000 A

Scanning electron microscope (SEM)

5-10 nanometers (nm)

50-100 A

Transmission electron microscope (TEM)

0.5 nanometers (nm)

5 A

Bright-field microscope

Advantages: convenient, relatively inexpensive, widely available oDisadvantages: resolving power 0.2 micrometers at best can recognize cells but not fine details needs contrast; cells are mainly water and don't contrast with their medium Easiest way to view cells is to fix and stain Fixation

preserves cells; disrupts proteins, prevents decay/degradation typical treatments: heat, formalin, glutaraldehyde

Staining Simple Stains

adds colored compounds -----contrast

basic dyes: e.g. methylene blue, crystal violet. Cations ( + charges) bind to - charge groups on proteins, nucleic acids acidic dyes: e.g. eosin, acid fuchsin. Anions ( - charges); bind to + charges on proteins, phospholipids

Differential Stains allow differentiation between different organisms Examples: Gram stain Spore stain

Phase contrast microscope

Cells are mostly water, very little contrast from surrounding medium, so not very visible in light

Phase scope converts slight differences in refractive index and cell density into variations

Scope uses annular stop below condenser: thin transparent ring in opaque disk ----- hollow light cone

As light passes through specimen, some rays are deviated and retarded by ¼ wavelengthHave phase plate in objective lens: transparent optical disk with phase ringUndeviated light passes through ring, is advanced by ¼ wavelength bright background Deviated light doesn’t pass through phase ring, is not advanced.When light gets focused, deviated rays cancel out with undeviated rays, producing dark image where objects were

Advantage: can see live material without staining

Dark field microscope

oFluors are chemicals that adsorb light to produce excited electrons, later reradiate light = fluorescence

- need filters to remove this light from light traveling to ocular lens- only fluoresced light emitted from object will then appear to eye - need dark field condenser to create dark background - can couple flour to specific probe molecules (usually antibodies) bind to preparation- if sample is illuminated with wavelength of exciting light, then filter out that wavelength to prevent reaching the sample and nothing is seen - but if fluorescence occurs, different wavelength of light is produced, object is seen- good technique to detect specific microbe in complex sample. (e.g. detect gonococcus in vaginal smear)- requires correct microscope, fluors, technical skill

Fluorescence microscopy

Scanning electron microscopy

Differential Interference Contrast (DIC) Microscopy

Uses a polarizer to create two distinct beams of polarized lightGives structures such as endospores, vacuoles and granules a three- dimensional appearanceStructures not visible using bright- field microscopy are sometimes visible using DIC

The atomic force microscope (AFM) or scanning force microscope (SFM) is a very high-resolution type of scanning probe microscopy, with demonstrated resolution of fractions of a nanometer, more than 1000 times better than the optical diffraction limit.

developed by Gerd Binnig and Heinrich Rohrer in the early 1980s, a development that earned them the Nobel Prize for Physics in 1986

is one of the foremost tools for imaging, measuring and manipulating matter at the nanoscale

Confocal Scanning Laser Microscopy

Uses a computerized microscope coupled with a laser source to generate a three-dimensional image

Computer can focus the laser on single layers of the specimen

Different layers can then be compiled for a three-dimensional image

Resolution is 0.1 um for CSLM

RELATIONSHIP OF STRUCTURE TO FUNCTION

The ratio of these two numbers is 4.2 x 108 !!! Eucaryotes span an even larger range of about 1018!!

SIZE

the difference between an average bacterium (measured in mm) and an elephant (measured in meters) V = 4/3 r3 Volume of sphere/ coccus V = r2 h Volume of cylinder/ bacillus

smallest bacteria (e.g., mycoplasmas) 0.2 mm in diameter--- V = 4.18 x 10-15 cm3 largest bacterium known (60 mm x 600 mm--- V = 1.74 x 10-6 cm3

Surface of cylinder S = 2 r h, hence S/ V = 2/r Surface of sphere S = 4 r h, hence S/ V = 3/r

As r gets smaller and smaller, S/ V gets larger and larger

-- difference between sphere and cylinder becomes insignificant

If elephant is approximated by a sphere of 3 m, then 3/3 = 1.0 m-1 Correspondingly,

-- a small mycoplasm would have S/ V = 3/1 x 10-7 = 3 x 107 m-1

Methods for Measurement of Cell Mass

1. Direct physical measurement of dry weight, wet weight, or volume of cells after centrifugation.

2. Direct chemical measurement of some chemical component of the cells such as total N, total protein, or total DNA content.

3. Indirect measurement of chemical activity such as rate of O2 production or consumption, CO2 production or consumption, etc.

4. Turbidity measurements employ a variety of instruments to determine the amount of light scattered by a suspension of cells.  Particulate objects such as bacteria scatter light in proportion to their numbers. The turbidity or optical density of a suspension of cells is directly related to cell mass or cell number, after construction and calibration of a standard curve. The method is simple and nondestructive, but the sensitivity is limited to about 107 cells per ml for most bacteria.

Methods for Measurement of Cell Numbers

1. Direct microscopic counts are possible using special slides known as counting chambers. Dead cells cannot be distinguished from living ones.  Only dense suspensions can be counted (>107 cells per ml), but samples can be concentrated by centrifugation or filtration to increase sensitivity.

2. Electronic counting chambers count numbers and measure size distribution of cells. Such electronic devices are more often used to count eukaryotic cells such as blood cells.

3. Indirect viable cell counts, also called plate counts, involve plating out (spreading) a sample of a culture on a nutrient agar surface.

Table 1. Some Methods used to measure bacterial growth

Method Application Comments

Direct microscopic countEnumeration of bacteria in milk or cellular vaccines

Cannot distinguish living from nonliving cells

Viable cell count (colony counts)

Enumeration of bacteria in milk, foods, soil, water, laboratory cultures, etc.

Very sensitive if plating conditions are optimal

Turbidity measurementEstimations of large numbers of bacteria in clear liquid media and broths

Fast and nondestructive, but cannot detect cell densities less than 107 cells per ml

Measurement of total N or protein

Measurement of total cell yield from very dense cultures 

Only practical application is in the research laboratory

Measurement of biochemical activity e.g. O2 uptake CO2 production, ATP production, etc.

Microbiological assays

Requires a fixed standard to relate chemical activity to cell mass and/or cell numbers

Measurement of dry weight or wet weight of cells or volume of cells after centrifugation

Measurement of total cell yield in cultures 

Probably more sensitive than total N or total protein measurements

Micrometry - measurement of minute objects with a micrometer.

Calibration Factor - actual distance between any two adjacent lines of the ocular micrometer by observing how many lines of the stage micrometer (Sm) are included within a given number of lines on the ocular micrometer (Om).

The distance between any two adjacent lines on the stage micrometer is = 0.01 mm (10 microns)

C. F. = Sm x 0.01 mm (10 microns) Om

Example : 10 divisions in Om match with 6 divisions in Sm

6 x 0.01 mm = 0.006 mm 10

or 6 x 10 µm = 6 µm 10

10 = deca

102 = hecto

103 = kilo

106 = mega

109 = giga

1012 = tetra

10-1 = deci

10-2 = centi

10-3 = milli

10-6 = micro

10-9 = nano

10-12 = pico

10-15 = femto

10-18 = atto

The Sleeve does not move.   It looks like a ruler with ten numbers.   The space between each number is divided into quarters.   As the Thimble rotates around this Sleeve it covers up, or reveals the numbers marked on the Sleeve.

It is easy to read a micrometer if you think of the markings on the Sleeve as dollars and quarters.

What are the readings on the micrometers as shown?

$2 or .200

As the thimble rotates, you add those pennies to the dollars and quarters

References:

Brock Biology of Microorganisms Internet Sources (especially most of the diagrams)