Transcript Methods for Measurement of Cell Numbers

Analytical Microbiology
-
analytical instrumentation in microbiological research
and applications which include
Gas chromatography
GC- mass spectrometry
HPLC/Ion chromatography
RAPD
Electrophoresis
FA analysis/Quinolone Analysis
PCR
TLC
Miscroscopy
- 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)
- using a simple microscope, he was the first
to observe microorganisms.
Animalcule
Leeuwenhoek’s drawings of bacteria
A, C, F and G are rod –shaped
E – spherical or coccus-shaped, H cocci packets
Hooke’s observation laid the groundwork for development
of the cell theory, the concept that all living things are
composed of cells.
Cell walls in cork tissue
Born on July 18, 1635
Robert Hooke (1665) - observed that plant
material was composed of little boxes, he
introduced the term “ cell.”
Drawing of Robert Hooke, which represents one of the first microscopic descriptions
of microorganisms
A 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.
2.
3.
Ocular/eyepiece
Objectives
Condenser
LPO =
HPO =
OIO =
Scanner
10x (100)
40/45x (450)
100x (1000)
= 4x (optional)
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
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
Dissecting microscope
Compound microscope
Coxial binocular microscope
Inverted microscope
Inclined microscope
Zoom tinocular
microscope
Projection microscope
Microscope with cleaning
kit
Research projection
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
Human eye
Resolving Power
0.2 millimeters
(mm)
RP in
Angstroms
2,000,000 A
Light microscope
0.20 micrometers
(µm)
2000 A
Scanning electron
microscope (SEM)
5-10 nanometers
(nm)
50-100 A
Transmission electron
microscope (TEM)
0.5 nanometers
(nm)
5A
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
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
Phase contrast microscope
As light passes through specimen, some rays are deviated and
retarded by ¼ wavelength
Have phase plate in objective lens: transparent optical disk with
phase ring
Undeviated 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
-
-
Fluorescence microscopy
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
Scanning electron microscopy
Differential Interference Contrast
(DIC) Microscopy
Uses a polarizer to create two
distinct beams of polarized light
Gives structures such as endospores,
vacuoles and granules a threedimensional appearance
Structures not visible using brightfield 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
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
The ratio of these two numbers is 4.2 x 108 !!!
Eucaryotes span an even larger range of about 1018!!
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 count
Enumeration 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 measurement
Estimations 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
Microbiological assays
uptake CO2 production, ATP
production, etc.
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
Probably more sensitive
than total N or total protein
measurements
Measurement of total cell
yield in cultures
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
deca
10-1
= deci
10-2
= centi
10
=
102
=
hecto
10-3
= milli
103
=
kilo
10-6
= micro
106
=
mega
10-9
= nano
109
=
giga
10-12
= pico
1012
=
tetra
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)