counting method of microbes

1. Counting Procedures

2. Determination of Bacterial numbers
Difficult to count in numbers from mixed culture.
Enumeration techniques must be chosen with care.
Methods used for one group is different from those
for other groups.
Special techniques used for specific physiological
groups that requires special sampling and processing
procedures.
No universal method can be applied to all microbes
and all habitats so a variety of methodological
approaches.
Direct approach and indirect (for viable count)
approach

3. Direct count procedure
Microscopic count: Gives the highest estimates of
numbers and can be used indirect measure of
biomass.
Some disadvantages are there
Cannot distinguish live and dead cells,
Too much background debris may cause
underestimation
Inability to perform further studying

4. o Counting chamber like hemocytometer, Petroff-Hauser
chamber can be used for counting large cells.
o A modified agar film technique for fungi count.
o A thin dried agar film on slide stained with phenolic
aniline blue. Length of mycelia can be measured.
o The mycelia may be hidden in soil particles so proper
grinding is necessary.
o The agar film technique when combined with
fluorescence microscopy using fluorescein diacetate to
stain living fungi for biomass measurements.
o One problem is high background fluorescence due to
esterases that reacts with the dye.

5.  Acridine orange, DAPI (4,6diamidino-2-phenylindole),
FITC for epifluorescent microscopy direct counting of
bacteria
 DAPI is better than Acridine orange for counting (stays
constant for 24wks).
 AO-stained count decreases after 4 weeks of storage.
 Bisbenzimide binds to adenine and thymine rich areas
and increases fluorescence of DNA. One advantage of
using this is detergents or other biological materials
do not affect DNA.
 Low-fluorescing immersion oil is important to
minimize background autofluorescence.
• Polycarbonate filters are superior to cellulose filters
for direct counting, uniform pores and flat surface. So
retains all the bacteria.

6. • Filters can be dyed as a dark background and
fluorescent microbes can be counted.
• If AO stained then green fluorescence indicates
higher DNA conc. Orange-red fluorescent indicates
cells richer in RNA and proteins.
• DAPI gives intense blue fluorescent with DNA.
• Counts by direct epifluorescent microscopy is two-
fold higher than cultural techniques.
• Direct counts are often directly proportional to
biomass and so can be used to calculate biomass.
• Direct count by epifluorescent microscopy is
applicable to all types of habitats for numbers of
microorganisms even though great differences in
population sizes and physiological types.

7. From frequency of dividing cells (FDC) in situ growth-
rate can be also measured that correlates with other
measures of growth.
 its not always valid and its difficult to observe
dividing cells by light microscope.
Fluorescent antibody technique can help in
enumeration.
It is very specific for an individual microbial species.
FITC (fluorescein isothiocyanate) a fluorochrome is
conjugated with the antibody.
Monoclonal antibodies have been used
• Ex. Methanogens.
Some disadvantages is sometimes background
nonspecific fluorescent may occur. Highly specific so
different strains of same sp. maybe precluded,
possible reactivity with different organisms.

8. • Direct immunofluorescence has been used for
detecting and counting bacteria from water, in
buried slides, on root surfaces, nodules etc.
• Viable but nonculturable bacteria in aquatic or
marine. Ex. V. cholerae
• ELIZA has been used in peat and soil.
• Fluorescent ELIZA can be compared with antibiotic
resistance technique.
• Fluorescent gene probes has been used for
counting specific populations. Probes are for
specific diagnostic genes. Mostly targets ribosomal
RNA can permit single cell detection.

9. • Multiple populations can be counted in the same sample using
different gene probes each conjugated with a fluorescent dye of
different color.
• Visualized by scanning confocal laser microscopy.
• Sensitivity of the gene probe detection can be enhanced by digital
image analysis and confocal laser microscopy.
• These analyses can be used for in situ studies on gene structure-
function relations.
• Fluorescent labeled gene probes together with antibodies have
been used in mixed cultures.
• Counter staining with DAPI and using scanning confocal laser
microscopy or epifluorescence microscopy with CCD (charge
coupled device) camera permits exact location of individual cells.
• The combined application of fluorescently labeled gene probes and
antibodies is a powerful tool for monitoring specific bacterial strains
in the environment.

10. • Epifluorescent microscopy can be converted to
automatic data analysis when large number of
samples present.
• Counts done by standard visual analysis and by
image analysis gave statistically equal results.
• For good strong image analysis the staining must
be very good with almost no background
fluorescence.
• So extreme care is required.
• Highly sensitive camera is required.
• Camera should also be able to distinguish cells and
debris.
• Although expensive but less time reqd. and
increased productivity.

11. • Fluorescent label in combination with other
procedure gives total count and living cells too.
• AODC and INT(2-[p-iodophenyl]-3[p-nitrophenyl]-
5-phenyl tetrazolium chloride) stain that can
detect respiring cells. Respiring cells reduce INT to
INT formazan and living cells accumulate
intracellular dark red spot.
• Numbers of Actively growing bacteria in presence
of Nalidixic acid that inhibits cell division by Direct
Viability Count (DVC). Cells elongate but no cross-
wall formation so does not divide. Actively
growing cells can be differentiated from normal or
dead cells. Time of incubation is important.

12. • No single method is accurate for direct viable count.
• Each method measures a different characteristic of the
cell.
• So its important to use more than one method to
determine viability in environmental samples.
• Many microbes though viable remains non-culturable.
• Autoradiography and direct microscopy can be done to
get bacterial numbers of actively metabolizing cells.
Bacteria growing on radioactively labeled substrate
collected on filter that is placed on a glass slide and
coated with a photographic emulsion. Active cells will
have dark silver grains surrounding the cells.
• Scanning Electron microscopy can be used in place of
light microscopy and is comparable to epifluorescent
microscopy.

13. • Particle Count Method:
• Coulter counter can be directly used to count cell
numbers.
• It electronically measures particle numbers of
fixed size range.
• Separate size range for bacteria or protozoa.
• Disadvantage is it also counts small nonliving
particles.
• Flow-cytometry helps in recognition of microbes
has been developed together with the colony
counter.

14. • Viable Count Procedures
• Two approaches
• 1. Plate count technique
• 2. Most probable number (MPN)
• For all viable count the organisms must be
separated into individual entities.
• Viable count procedures are selective that varies
with the particular count procedure.
• Selectivity is a disadvantage as it cannot estimate
total but only selective group.
• Only live cells will be recovered and grow in the
laboratory and detected.

15. • Plate Count: Selective and Differential Viable .
Culture Methods
• Best use by Agar plate count. But sometimes results
are misinterpreted as never total count can be
obtained.
• Various media and incubation conditions are
employed
• Agar is the solidifying agent.
• Plates can be streaked, spread and poured.
• Diluted samples are spread on agar or mixed with
molten agar to pour on plates.
• Care to keep microbes alive during plating.
• Oxygen, temp etc to be taken care of.
Silica gel used instead of Agar for solidifying Plates.
Sources of nutrition like C, S, P, N and Fe, M, Na Ca, Cl,
etc

16. • Media composition, time of incubation, Incubation
temperature are main consideration.
• Media must meet the nutritional requirements
including growth factors. Sometimes conc. is toxic.
• Obligate anaerobes are grown on roll-tubes an
extension of pour plates.
• The tubes are incubated until colonies develop
when they are counted.
• An advantage of viable count is growth conditions
can be adjusted so that only members of a defined
group are enumerated.
• Selective and Differential plates

17. Selective plate count favors desired group.
Other groups can be avoided by media
composition or incubation conditions.
Differential count includes all members but desired
group can be permitted by some distinguishing
characters.
Media can be designed for the selective
enumeration of fungi specially nonfilamentous
fungi and spores
Suitable for yeast count.
Bacterial inhibitors (dye rose bengal or antibiotics
neomycin, streptomycin) are added to the plates to
make them selective.
Or lowering pH of media.

18. Reagents added to media makes them differential.
It allows immediate visualization and
differentiation of the desired bacteria.
Reagents may be added after incubation to detect
desired bacteria.
The advantage of this is that it permits one to
distinguish the microbes being enumerated and at
the same time visualize others in the sample.
EMB and McConkey’s agar are both selective and
differential.
Selective for Gram negative. Differentiated by their
capability of lactose utilization gives colored
colonies.