http://www.sciencedirect.com.libproxy1.nus.edu.

sg/science/article/pii/S002197971731267
5
Synthesis and characterization of alginate beads
encapsulated zinc oxide nanoparticles for bacteria
disinfection in water
2.4. Antibacterial studies
To determine the antibacterial activity of the alginate nanocomposites, gram positive
(Staphylococcus aureus, ATCC 25923) susceptibility was investigated through batch
experiments. The bacterial strains were obtained from the CSIR Environmental Laboratory
(Pretoria, South Africa) and were re-cultured using Mueller-Hinton broth and agar according
to the manufacturer’s instructions. In brief, bacteria was grown on Mueller-Hinton agar plates
and incubated at 36 ± 2 °C for 24 h. From these plates, one loop of bacteria was grown in
100 ml of sterile Mueller-Hinton broth and incubated in a shaking incubator (FMH Incubator
Shaker, ISKO 160) at 150 rpm for 24 h. The bacteria were harvested through centrifugation at
4000 rpm for 15 min and washed twice with sterile 0.01 M PBS (pH 7.4). Stock solutions of
the bacteria were prepared by re-dispersing the final pellets in 10 ml of the PBS solution.
From this bacteria suspension, serial dilutions were done using sterile saline water (0.9%
w/v) to obtain bacteria solutions with known concentrations. Three different concentrations
of synthetic bacteria contaminated water, 200 cfu/ml, 700 cfu/ml and 1 000 cfu/ml were
prepared using sterile normal saline water. Sterilized bottles were filled with 100 ml of the
synthetic water and the alginate nanocomposites were added. To evaluate the minimum
amount of the nanocomposites that will be required to inactivate the bacteria, the
nanocomposites were weighed in 0.2 g, 0.5 g and 1.0 g. The bottles were shaken at 36 ± 2 °C
in a water-bath shaker for 24 h at 150 rpm. At different time intervals, 1 ml was withdrawn
and added into micro test tubes containing 100 µl of sodium thiosulphate. The aliquots were
vortexed and plated on agar plates without any further dilutions. Incubation lasted for 24 h at
36 ± 2 °C. The colonies formed after incubation were counted, which represented viable
bacteria during the set sampling intervals. The antibacterial evaluation was done in triplicate
and the averaged are reported.

http://www.sciencedirect.com.libproxy1.nus.edu.sg/science/article/pii/S004896971731311
6
Evaluation of synergy and bacterial regrowth in
photocatalytic ozonation disinfection of municipal
wastewater
2.3. Experimental equipment and process description
Two experimental systems were employed in this study: (i)
ultraviolet (UV) photocatalytic ozonation system; and (ii) solar
(SL) photocatalytic ozonation system.
2.3.1. Ultraviolet photocatalytic ozonation process
Experiments were carried out in an annular quartz
photochemical reactor with a capacity of 0.7 L which was
wrapped in aluminium foil and operating in a closed
recirculation system (Fig. 1). It was equipped with a medium
pressure mercury lamp (Heraeus TQ 150 W), placed axially in
a quartz immersion tube. A cooling system for the lamp was
provided by circulating water which allowed the temperature to
be controlled below 30 °C. The lamp emission spectra had a
dominant peak at 366 nm. The light intensity of the lamp was
measured using a Goldilux Smart Radiometer (MIT Pty Ltd.,
SA) and found to be 70 mW/cm2. The feed water was pumped
into the reactor and the catalyst added. The mixture was stirred
magnetically for 30 min in the dark to attain adsorption-
desorption equilibrium and thereafter, the UV lamp was
switched on. Ozone was produced onsite using dry
compressed air in an ozone generator (Wassertec, Light Blue
ozone generator). Ozone was bubbled into the bottom of the
reactor by means of a pipe diffuser. The water was
continuously re-circulated between the feed tank and the
reactor and at regular time intervals water samples were
collected through the sampling valve into sterile bottles for
quantification of the bacterial concentrations. All experiments
were performed at the natural pH of the wastewater.
Throughout the experiments the ozone in the off gas was
captured and passed through a gas absorption flask containing
KI as per the standard procedure (APHA et al., 2005). Three
control experiments were done: (i) using the photocatalysts
without UV light, (ii) using UV light without the photocatalysts,
and (iii) using the doping metals (Ag, Cu and Fe) at the
concentrations used for doping TiO2.
 Download high-res image (171KB)Download full-size image
Fig. 1. Process flow diagram for the UV photocatalytic ozonation unit (Mecha
et al., 2016a).
2.3.2. Solar photocatalytic ozonation process
The solar photocatalytic ozonation set-up is illustrated in Fig. 2.
The solar reactor was tubular, made of transparent borosilicate
glass (1 m in length and 0.04 m in diameter). A parabolic trough
collector was used to reflect solar radiation onto the reactor.
The average intensity of solar radiation was measured using a
Goldilux Smart Radiometer (MIT Pty Ltd., SA) and found to be
37.6 mW/cm2 over a range of visible light wavelengths (400–
700 nm). The feed water was pumped into the reactor under
dark conditions (reactor covered using an opaque sheet) and
the catalyst added. The mixture was stirred magnetically for
30 min in the dark to attain adsorption-desorption equilibrium
and thereafter, the reactor was uncovered and exposed to
sunlight. Ozone was produced onsite using dry compressed air
in an ozone generator (Wassertec, Light Blue ozone
generator). Ozone was bubbled into the reactor by means of a
pipe diffuser. The water flowed through the reactor and back to
the feed tank where it was again re-circulated using a pump.
Experiments were conducted on sunny days between 10:00
and 15:00 local time. At regular intervals, samples were
collected through the sampling valve into sterile glass bottles
and treated as described in Section 2.3.1. Control experiments
were done using: (i) the photocatalysts without light and (ii)
solar light without the photocatalysts.

Download high-res image (157KB)Download full-size image

Fig. 2. Process flow diagram for the solar photocatalytic ozonation unit (Mecha
et al., 2016a).
2.3.3. Evaluation of bacterial regrowth
To evaluate bacterial regrowth, samples form the disinfection
experiments (ozonation, photocatalysis and photocatalytic
ozonation) for which no bacteria were detected after treatment
were selected. For ozonation, bacteria could not be detected in
the treated samples after 45 min of disinfection; while for
photocatalysis, complete disinfection was attained after 60 min;
for UV photocatalytic ozonation, no bacteria were detected in
the samples after 15 min, and for solar photocatalytic
ozonation, complete disinfection was attained at 30 min. All
these samples were kept for in the dark at ambient temperature
(25–30 °C) and post-disinfection monitoring was done to
determine the survival and regrowth of the target bacteria after
24 and 48 h.
2.4. Physicochemical analysis of treated wastewater samples
The chemical oxygen demand (COD) was determined using the
Closed Flux Colorimetric method (APHA et al., 2005). Turbidity
was measured using a 2100N laboratory Turbidimeter (Hach,
USA). The pH was measured using an HQ30d portable pH
meter (Hach, USA). The DOC of the samples was measured by
a TOC-L CPH Total Organic Carbon analyser (Shimadzu,
Japan). The concentrations of phosphate and nitrate were
evaluated using standard methods (APHA et al., 2005).
2.5. Disinfection kinetics and modelling of survival curves
The bacterial survival was predicted using the Chick-Watson
disinfection model. The model's general expression based on
the Chick (1908) and Watson (1908) models is as follows:
(1)
where No and N are the number of bacteria (CFU/mL) at the
start of the process and at a time t, respectively, k is the
disinfection rate constant, c is the concentration of the
disinfecting agent at time t, and n the reaction order.
In photocatalytic ozonation processes, the concentration of the
disinfecting agent could be considered to be constant with time
for a given catalyst concentration, irradiation source and ozone
dosage. Consequently, in this case the equation could be
rewritten as follows:
(2)
where k′ is the concentration-independent rate constant
(min− 1). This model was used to fit the photocatalytic
disinfection data.
To quantify the effectiveness of the combined process, the
synergy index (SI) was calculated for photocatalytic ozonation
using the following equation (Mecha et al., 2016a):
(3)
where LRV is the log reduction value and the subscripts
represent ozonation (Oz) and photocatalysis (Phot).
To evaluate the reactivation of bacterial cells after 24 and 48 h
retention in the dark, the percentage degree (D) of reactivation
or decay was quantified using the method described by
previous investigators (Li et al., 2013):
(4)
where Nr (CFU/mL) is the concentration of the bacteria in
treated water after 24 or 48 h storage in the dark. Eq. (4) shows
the percentage of repaired bacteria among bacteria inactivated
by photocatalytic ozonation disinfection. If the value of D is
negative, then it is termed the degree of decay.
2.6. Photocatalyst recovery and reuse
After the completion of the disinfection process, the
photocatalysts were allowed to settle in the reactor for 30 min
and then the supernatant was poured. The photocatalysts were
collected and centrifuged to separate the particles from the
remaining wastewater. The photocatalysts were washed three
times using deionised water and dried in an oven at 100 °C for
2 h, thereafter they were reused.

http://www.sciencedirect.com.libproxy1.nus.edu.sg/science/article/pii/S004313541730784
4
Electrochemical disinfection of bacteria-laden water using
antimony-doped tin-tungsten-oxide electrodes
2.4. Disinfection experiments
The coated electrode was sonicated in deionized water for 20 min before experiments to
remove residual contamination from the surface of the electrode. The reactor was operated in
a three-electrode mode and potentials were noted relative to SCE. The disinfection of
bacterial suspension was carried out at constant current densities of 0.2, 1, 2, 4, 6 and
10 mA/cm2 with 107 CFU/mL initial bacterial concentration. The voltage and temperature at
each sampling time were recorded. Before starting each electrochemical experiment, a
100 μL sample was taken from the reactor with a sterile pipette to determine the initial
concentration of bacteria. Also, samples were collected during the runs at selected time
intervals and were analyzed as described below.

2.5. Bacterial sample analysis

The drop plate method was used to estimate the concentration of viable bacteria in the
samples by enumerating the CFU grown on a LB agar plate (Herigstad et al., 2001). The
samples taken from the bacterial suspension in the reactor were serially diluted down to
10−5 (with a factor of 10−1) of its initial concentration using the background electrolyte. Each
dilution was thoroughly mixed, from which the 10 μL drops were dispensed over the one-
sixth of the surface of an agar plate which was then incubated at 37 °C for 14 h. After this
time, CFU were counted and the average of the total count of CFU over 3 drops at the
countable dilution was determined to calculate the initial concentration of bacteria. The
viable cell density was initially 107 CFU/mL in disinfection tests.

http://www.sciencedirect.com.libproxy1.nus.edu.sg/science/article/pii/S246820391730056
0#bib42
Zinc oxide nanoparticles for water disinfection
5.2. Methods to measure sensitivity of microorganism to
antimicrobial agents
Different methods are used to assess antibacterial activity
in vitro. Antibacterial tests are usually done in aqueous media
or cell culture media. In these methods, the culture media,
trypticase soy broth, Luria–Bertani broth, nutrient agar, tryptic
soy agar, were accordingly selected and autoclaved. The
stocks of ZnO nanoparticles suspensions are also usually
prepared, and serially diluted to different concentrations [10].
The antimicrobial activity of ZnO nanoparticles is measured
qualitatively using the disk diffusion test and quantitatively by
minimum inhibitory concentration (MIC) assay using standard
microbial methods [42].
The most common method for evaluating in vitro activity is
through agar disk diffusion method, owing to its simplicity and
low cost. In this method, agar plates are inoculated with a
standardized inoculum of the test microorganism. Then, a
paper disk, containing the antibacterial solution at a desired
concentration, is placed on the agar surface. The petri dishes
are incubated under suitable conditions. The antimicrobial
agent diffuses into the agar and inhibits growth of the
microorganism. Then the diameter of the zone of inhibition
(ZOI) is observed around each disk, the size of which will
depend on the sensitivity of the organism to the antimicrobial
agent, and measured [26,43].
Bacteria growth curves show the relationship between microbial
population as expressed by optical density (OD) or absorbance
measured at 600 nm versus time of incubation. Fig. 3 from the
studies of Navale et al. [18] and Jones et al. [32] shows that
higher concentrations of ZnO nanoparticles retarded the growth
of the pathogen Staphylococcus aureus and B. subtilis.
 Download high-res image (180KB)Download full-size image
Fig. 3. Bacteria growth curve of Staphylococcus aureus in the presence of
ZnO.
The colony-forming unit (CFU) is a measure of viable bacterial
or fungal cells. After culturing the microbes and treating with the
antibacterial agent, the growth inhibition is determined by
counting the number of CFUs. Xie et al. [44] evaluated the
bactericidal activity of ZnO nanoparticles against common food
pathogens Campylobacter jejuni and E. coli. As shown in
Fig. 4, the C. jejuni cells were killed after 1.5 and 3 h at
concentrations of 0.5 and 0.1 mg mL−1, respectively. The graph
also shows the susceptibility of C. jejuni compared to E.coli.

Download high-res image (180KB)Download full-size image

Fig. 4. Antibacterial activities of ZnO nanoparticles against C. jejuni and E. coli
at different ZnO concentrations (Xie et al. [44]).
The inhibition efficiency is defined as
(1)
where N1 and N2 are the number of colonies on the plates
before and after inhibition, respectively. Fig. 5 shows the
inhibition efficiency of ZnO at different concentrations against
E. coli as investigated by El Saeed et al. [15] and Padmavathy
and Vijayaraghavan [9].
 Download high-res image (130KB)Download full-size image
Fig. 5. Inhibition efficiency of different ZnO concentrations against E. coli.
The MIC gives a quantitative assessment of the potency of an
antibiotic. It is the lowest antimicrobial concentration that
inhibits the visible bacterial growth in vitro[42].
5.3. Factors affecting antimicrobial activity
Antibacterial activities of nanoparticles depend on two main
factors: a) physicochemical properties of nanoparticles, and b)
type of bacteria [37]. Although dosage is an important factor,
particle size, particularly in the nanoscale, is a more important
determinant of antibacterial activity than dosage. Accordingly,
the toxicity of metal oxides such as ZnO does not necessarily
depend on the internalization of the nanoparticles in the
bacteria. These nanoparticles can also change
microenvironments near the bacteria and produce ROS or
increase the nanoparticles solubility leading to bacterial
damage [29].
http://www.sciencedirect.com.libproxy1.nus.edu.sg/science/article/pii/S095656631630129
4
Bacteria counting method based on polyaniline/bacteria
thin film
2.2. Bacterial cultivation and cytometry
Microbial strains of Bacillus subtilis, Escherichia coli (BL-21)
and Streptococcus thermophiles were obtained from School of
the Environment and Safety Engineering. B. subtilis and E. coli
were cultured in 125 mL of Luria Bertani broth at 37 °C for 20 h,
and S. thermophiles cultured in MRS Broth. The bacterial cells
were centrifuged for 10 min at 3500 ɡ and washed three times
with sterilized water at 4 °C. Finally, bacteria were re-
suspended in water under gentle vortex mixing. The count of
bacteria in the suspension was obtained by plate colony-
counting method. The re-suspended bacteria suspension was
diluted with water to obtain needed suspension of different
concentration before use.
2.3. Deposition of PANI film on bacteria anchored GCE
Prior to modification, the GCE was first polished with sand
paper followed by 1.0-, 0.3-, and 0.05-μm alumina slurry. After
successive sonication in ethanol and double-distilled water, the
electrode was rinsed with double distilled water and allowed to
dry at room temperature.
As illustrated in Scheme 1, 10 µL of bacteria suspension of
different concentration was dropped onto the preprocessed
GCE, and dried for 15 min at 50 °C to immobilize the bacteria
cells on the surface of the GCE. By cyclic voltammetry (CV)
technique, the potential of the bacteria anchored GCE was
scanned between –0.2 and 0.9 V at 50 mV/s for 10 cycles in a
solution of 0.1 M aniline containing 0.5 M H2SO4 to deposit a
bacteria decorated PANI (PANI/bacteria) film. The temperature
of the solution was controlled at 0 °C using ice-water bath.
Finally, the PNAI/bacteria film modified GCE was thoroughly
rinsed with water.

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Scheme 1. Schematic for the preparation and characterization of
PANI/bacteria film on GCE.
2.4. Electrochemical measurements
The prepared PANI/bacteria GCE was characterized with CV
and electrochemical impedance spectroscopy (EIS) technique
in 0.5 M H2SO4 at 0 °C using ice-water bath. The CV tests
were performed between –0.2 and 0.9 V at 50 mV/s. For EIS
tests, the AC voltage amplitude was 5 mV, the applied potential
was 0.23 V, and the frequencies ranged from 10 kHz to
0.01 Hz.

http://www.sciencedirect.com.libproxy1.nus.edu.sg/science/article/pii/S095671351530203
6
The role of heterotrophic plate count bacteria in bottled
water quality assessment
http://www.disknet.com/indiana_biolab/b038.htm

How to Count Bacteria

Counting Bacteria by Dilution and Plating
The example below will help you understand how to dilute and plate
bacteria. This example is the way that I usually set up my tubes for
assaying the bacteria titer when I am working with log-phage E. coli
B in phage experiments. I suggest you print a copy of this diagram
and use it at your work bench.
Notice that I transferred 0.1 mL from the bacterial culture into tube
#1. Tube 1 which contains 9.9 mL of Diluting Fluid (DF). Note that
0.1 mL + 9.9 mL = 10 mL. Therefore, Tube #1 contains a 100-fold
dilution of the original liquid culture. Tube #2 is handled exactly the
same way. For tube #3, 0.1 mL was diluted into 1.9 mL making this a
20 fold dilution. Notice I plated 0.1 mL from Tub #3 which is a 10
fold factor. Suppose I count 104 bacteria on the plant. Then the
calculation of titer of the original bacteria culture is 100 x 100 x 20 x
10 x 104 => 2.08 x 108 cells/mL.

The diagram above illustrates a typical dilution and plating. Here are
the details of each step:
Use a clean, sterile, dry pipet to remove 0.1 ml from the bacteria
sample and blow it into the 9.9 ml of dilution fluid in tube #1
and mix thoroughly by blowing lots of bubbles with the pipet
 for a couple seconds. Discard the pipet into the used jar for later
cleaning. Notice tube #1 now contains 1/100 the concentration
of bacteria in the original sample because 0.1 ml is 1/100 of 10
ml. Since nearly 0.1 ml of liquid may cling to the outside of the
pipet, you must wipe the pipet with Kleenex or toliet paper
before inserting the pipet into tube 1.
Using another clean, sterile, dry pipet remove 0.1 ml from Tube 1,
wipe pipet, blow contents of pipet into Tube 2, continue
blowing bubbles for a second or two for good mixing.
Using another clean, sterile, dry pipet remove 0.1 ml from Tube 2,
wipe, blow contents of pipet into Tube 3, continue blowing
bubbles for a second or two for mixing.
Plate 0.1 from Tube 3. See below for information on plating.
There are many types of pipets, we are using blow out pipets and
that is indicated by a frosted ring on the pipet at the top end.
Read my page on types of pipets.
Suppose 125 colonies grow in the petri plate. Multiply the dilution
factors by the count to obtain the titer of the bacteria sample: 100 x
100 x 20 x 125 ==> 250,000,000 bacteria per milliliter in the original
sample. 250,000,000 = 250 million = 2.5 x 108 bacteria per milliliter.
It is important to understand Scientific Notation which is explained on
page b038a.htm.

resume here
Beginners usually assume each colony grew from a single bacteria,
but experts prefer say each colony originated from a colony-forming
unit. Dead bacteria do not form colonies. Some bacteria occur as
single cells. Other species hang together in chains or clumps of 2 or
more bacteria. A piece of dirt with 10 bacteria on it will form a single
colony. Molecular biologists do everything with as much precision as
possible. Therefore, they prefer to work with bacteria which do not
form chains or clumps.
Other Methods for Counting Bacteria
Counting Chambers
Counting chambers automaticly fill with a certain volume. There are
several kinds of counting chambers but they all consist of a special
microscope slide with a coverglass.
 Fill chamber with bacterial liquid culture according to manufactuer's
instructions.
Place slide under microscope
Count the bacteria according to the instruction sheet.
Calculate the number of bacteria per milliliter of orginal sample
from the known volume of the counting chamber according to
the instruction sheet.
One disadvantage of this method is that you can't tell which bacteria
are alive. Therefore the count includes dead bacteria. Thus this
method is useless in disinfection studies.
Most Probable Number.
Dilute the bacteria so that each tube contains less than one bacteria
on average
Count the number of tube which grew
Using a special method calculate the Most Probable Number
to be continued and revised. page b038b will give more details on
the MPN method.
Membrne Filters
• Water is filtered on a filter which has holes smaller than bacteria
• The filter is then placed on a dish of agar and bacteria grow on the
top of the filter.
• Count the bacteria and calculate the cell titer.
One big advantage of this method is that huge volumes of water can
be filtered to show a few bacteria per liter. The filter method also
allows one to rinse the filter by following the sample with sterile
clean wter so that anything interfering with the growth of bacteria is
rinsed away.
Photometers and Spectrometers
• Light of a suitable wavelength is passed through a tube of the
culture.
• If there are lots of bacteria, less light gets through the culture.
• A meter reads the amount of light passing through the culture
• By consulting a standard curve which you have prepared by reading
the meter and plating the bacteria, you can estimate the number
of bacteria.
• This method is very useful in industrial fermentations where you
need to know the number of bacteria now rather than tomorrow
 after the plates have grown colonies.
How to Plate Bacteria
Pour plate or spreading plate.
hine light through the culture, cells will block light according to the
number of bacteria
cell counters move culture thru a hole and by conductance the cells
are counted. Cost tens of thousands of dollars.
Colony forming units are single bacteria or clumps of bacteria which
are able to form colonies on the medium used. 0.1 ml of the samle is
taken the pipet adjusted to zero and wiped with kleenex or toliet paper
and blown into tube #1. 0.1 ml into 9.9 ml is a 100-fold dilution. If
the titer of the sample was 10 to the eighth, it is 10 to the sixth in tube
#1.

http://delrio.dcccd.edu/jreynolds/microbiol
ogy/2421/lab_manual/numbers.pdf
COUNTING BACTERIA
http://morningsidemicro.wikidot.com/ways-
of-counting-bacteria
Ways Of Counting Bacteria
Microscope & hemocytometer:

1. What is a hemocytometer? How does a hemocytometer work?

It is a device that counts microscopic particles. The hemocytometer works becuase it creates a volumetric
grid in which is divided into many different sizes of cubes so that one can accurately count the amount of
particles in that cube and apply to the concentration of the entire sample.
Optical Density method
2. How does a spectrophotometer work to count bacteria?
-It doesn't directly count the bacteria or bacterial colonies, instead it measures the contents of a "colonized"
broth. Light is sent through a broth containing bacteria, and the amount of light that is able to shine through
determines the concentration of the amount of bacteria in the broth. The more light that shines through, the
less bacteria that are present, and vice versa.

Viable Count methods

3. What is the protocol for making a spread plate of a sample containing bacteria? How do you count the
bacteria on the plate? First, dilute the bacterial broth, than streak it out on a plate. Once incubated, than
one would count the colonies. Once one has the colony count, he or she can do some calculations to get
the bacterial count of the original sample
4. How is the protocol for a pour plate different from that of a streak plate? In a pour plate one would add a
ml of diluted bacterial broth which is poured out on a petri-dish. Then, 9 ml of agar medium is poured on
top. The contents are mixed and incubated. Then one would count the colonies and do some calculations
to find bacterial count.
5. Why would you use a pour plate rather than a streak plate? It seems easier to isolate specific colonies

Serial dilution of liquid samples

6. How can you count viable bacteria in a liquid sample when there is a high concentration of bacteria in
your sample? You would dilute the solution and then count the bacteria. Use the CV=CV equation to do
calculations based on the dilutions and bacterial count?

https://online.science.psu.edu/micrb106_wd
/node/6129

Counting Bacteria
Counting Bacteria
In order to talk about populations of bacteria, it's necessary to
count them. There are a number of ways to count microorganisms.
One of the most direct, (and tedious) ways to count bacteria is to
put them under a microscope and count them one at a time. This is
done to count Red Blood Cells for patients that have anemia and a
similar process can be used for bacteria. This will not tell you which
ones are alive and which ones are dead though. Also, it's possible to
take a culture and dry it out to make a powder. The dry weight can
then give an approximation of how many cells there are.
Another method that's often used is taking a measurement of
turbidity. When bacteria grow in a broth medium they divide rapidly
as we discussed. The more bacteria, the more cloudy the solution
becomes. Machines called Spectrophotometers commonly used in
chemistry labs can measure the turbidity or cloudiness of the test
tube. There are other machines, cleverly called Cell Counters, that
pass a very narrow stream of fluid through a laser beam. The
stream is so narrow the cells are essentially passing through in
single file. Each time a cell breaks the laser beam it's counted. The
problem with all of these methods is that none of them allow us to
easily count LIVING cells. There's no way to tell which cells are alive
and which are dead.
The most useful way to count living cells is to perform a Plate
Count or place a solution onto an agar plate and count the number
of colonies that form. Remember each colony represents a living cell
that multiplied into a big pile. The problem is that every milliliter of
media may contain billions or trillions of cells. No one wants to
count a billion colonies even if you could fit them all onto a single
plate. The solution is to Dilute the original sample a million times.
Then when media containing the bacteria is placed onto the plate
you're only dealing with a hundred or so colonies. After that a bit of
math will be needed to calculate the original undiluted sample.