EXPERIMENT 5: Determination of the Number of Total Coliform

Organisms in a Water Sample

Definition of Coliform Bacteria Group

Coliform group bacteria composed of all aerobic or facultative anaerobic that are grams negative,
non-spore-forming, rod-shaped, and that produce acid and gas within 48 hours at 35°C in lactose.
Meaning and Importance of Coliform Bacteria
These organisms are not pathogenic and are the indicators of potential contamination in the waters
and show the fecal contamination. Because of that these bacteria are also known as “indicator
microorganisms”. Typhus, dysentery, cholera, intestine infections are the main observed diseases
that are caused by coliform bacteria. Coliform bacteria can be defined as indicator organisms. The
reason for the search of coliform bacteria in the water is preferred because they are easier, cheaper,
and shorter than pathogenic bacteria.
Coliform bacteria are in the family Enterobacteriaceae, present in the intestinal tract of numerous
organisms, and can be classified as:
A. Saprophytic (harmless) under normal conditions
1. Coliform bacteria
a. Escherichia
b. Citrobacter
c. Klebsiella
d. Enterobacter
2. Proteus
3. Pseudomonas
4. Alcaligenes
B. Pathogenic Bacteria
1. Salmonella
2. Shigella

The most important specie of the coliform bacteria is the Escherichia Coli. Escherichia Coli has the
2-4 μm length, 0.4-to 0.7 μm width and has the shape of flat, rounded, spherocylinder. Its movement
is slower than typhoid bacilli (Salmonella typhi) and it is facultative anaerobic bacteria with optimal
reproduction temperature of 35 ° C. They can grow easily and abundantly in general growth
mediums. They form homogeneously in the liquid medium and their cultures are smell like feces.

Coliform Sources
The main source of coliform bacteria is the feces of human and warm-blooded animals. Therefore,
sewage waters in settlement centers and contaminated liquid wastes and solid waste leachate are
important sources of coliform. There are some non-fecal examples of coliform: Klebsiella sp.,
Citrobacter sp., Enterobacter sp.
Membrane Filter Method
Although it is costly, membrane filter method is very much straight forward, quick and versatile.
Membrane filter discs provided are made of cellulose acetate and their pore size is 0.45 m ± 0.05 m,
smaller than the size of any known bacteria. When liquid samples are filtered with these, bacteria are
1
retained on the surface (viruses however are not retained). When the filter placed on a pad impregnated
with special media, organisms grow over the surface and form colonies. After incubation, we can count
the number of colonies formed and determined bacterial count in the sample. When applying this
method to sewage samples, sample must be diluted sufficiently so as to yield 20-80 colonies/disc. The
primary disadvantage of this method is the membrane filter discs’ cost.

Procedure:
Prepare M-Endo Agar Plates (as 15 mL Endo Agar medium in the petri plate) (one for each group).
Take a 100 mL of "potable" water sample (1-tap water, 2-drinking water and 3-diluted pound water
including coliform specie), filter it through a sterile membrane filter (MF) that has been placed in a
special pre-sterilized filter holder assembly.
Place the membrane filter over the Endo-Agar petri dish, close the lid of the petri dish and put it into
the 35 0C incubator for 24 ± 2 h (do not invert the dishes). Count the resulting colonies that show a
metallic sheen and report the number of total coliform organisms/100 mL.

This a single-step MF method can be used where there are not many background organisms and where
there is no toxicity problem which may suppress coliform organisms. Otherwise a two-step MF method
is used. In the first step, the filter retaining the microorganisms is placed on an absorbent pad saturated
with lauryl tryptose broth. After incubation for 2 h at 35 oC, the filter is transferred to an absorbent pad
saturated with M-Endo broth or an equivalent and incubated for 20-22 h. at 35oC. The two-step method
is especially used for chlorinated secondary or tertiary sewage effluent and for industrial waste
effluents.

Multiple tube fermentation method

Multiple tube fermentation is the one of the two most commonly used methods for total coliform
determination and is still being performed in public health laboratories and preferred method rather
than membrane filter method.

The multiple-tube fermentation technique is a three-stage procedure in which the results are
statistically expressed in terms of the Most Probable Number (MPN) with the help of a table or
tables prepared according to statistical data. The experiment is usually done with 15 tubes.
Multiple tube method include
Stage 1 " Presumptive Stage"
Stage 2 " Confirmed Stage"
Stage 3 "Completion Test"
In practical applications, the first two steps give the result. The completion test is performed to
remove all suspects about the results and obtain a 95% confidence level.
Implementation of the Presumptive Stage
In this laboratory session we will perform only Presumptive stage. In this phase, both glucose-
containing liquid medium and lactose-containing liquid medium can be used. The amount of water
to be placed in the tubes containing the media relies on the used water property. For drinking water,
10 ml is put for each of the five tubes. A more precise measurement can be done using ten tubes.
Generally, for the other waters (drinking water not included) 10, 1, 0.1 ml inoculations can be
performed for 5 tubes of each that can result a total of 15 tubes.
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*Generally, presumptive stage is carried out by using five tubes for each sample, but in this
laboratory session, we will use three tubes for each sample as totally 9 tubes (10, 1, 0.1 mL
inoculations).

Figure 5. 1. Inoculation scheme

The samples are thoroughly shaken about 25 times and the appropriate cultures are inoculated. The
tubes are shaken gently and placed on the racks and placed at 35 ± 0,5 ° C. The tubes were checked
after 24 ± 2 hours and results are recorded as (+) for gas formation and (–) for no gas.

Media to be prepared:
Phenol red glucose broth and phenol red lactose broth (LB containing 5 g/L glucose or lactose and
0.03 g/L phenol red)
Preparation of phenol red glucose broth:
- 3 g Beef
- 5 g Peptone
- 5 g NaCl
- 5 g Glucose

3
 - Phenol red
- 1000 mL water
Preparation of phenol red lactose broth:
- 3 g Beef
- 5 g Peptone
- 5 g NaCl
- 5 g Lactose
- 0.03 Phenol red
- 1000 mL water

Materials:
-Tap water with coliform specie (as a wastewater sample)
-Tap water with B. subtilis (as a wastewater sample)
-Tap water
-Drinking water
Tubes with lactose broth with durham tubes in each sample with different concentrations
Tubes with glucose broth with durham tubes in each sample with different concentrations
Each group will select two sample (tap water coliform specie and tap water or tap water with
coliform specie and wastewater). Dilute coliform added tap water and wastewater as shown in the
above figure. Two tubes will be used for each dilution. No need to dilute tap water.

Procedure:
Inoculate each of two media (lactose and glucose media) with coliform specie and B.subtilis with
different dilutions as shown in the above Figure.
- At first, phenol red glucose/ lactose broth media is prepared.
- First set for each sample; test tube with Durham’s tube contains 5 mL double strength
lactose/ glucose broth and 5 mL sample.
- Second set; test tubes with Durham’s tube contain 5 mL single strength lactose/ glucose
broth, 1 mL sample and 4 mL distilled water.
- Final set; test tubes with Durham’s tube contain 5 mL single strength lactose/ glucose broth;
0.1 mL sample and 4.9 mL distilled water.
- For each sample, 3 tubes are used like; Glucose-WW10, Glucose-WW1, Glucose-WW0.1
- For drinking water and tap water, there is no dilution.
- Use a control tube in which no sample or bacterial culture is inoculated.
Examine the fermentation tubes after 48 hr and after 5 days and note as follows:
NR for no reaction (if there is only turbidity (no color change and no gas production))
A for acid production ( if the culture color turned to yellow and no gas production)
B for base production (darker red color than control tube)
AG for acid and gas production (yellow color and gas accumulation in Durham tubes)

4
Also, MPN table is used for evaluation of experiment results.

5
For results and discussion part, these tables will be used.

Number of Positive Tubes

Water Sample MPN/ 100 mL

3 Tubes, 10 mL 3 Tubes, 1 mL 3 Tubes, 0.1 mL

Glucose-B

Glucose-C

Glucose- Tap Water

Lactose-B

Lactose-C

Glucose- Tap Water

10 mL sample 1 mL sample 0.1 mL sample

Water Sample (Source)

NR A B AG NR A B AG NR A B AG

Glucose-B

Glucose-C

Glucose- Tap Water

Lactose-B

Lactose-C

Glucose- Tap Water

6
EXPERIMENT 6: MICROBIOLOGY OF ACTIVATED SLUDGE
Objective: Defining microorganisms in activated sludge and soil sample, observing under
microscope and isolation of specific bacterial types from mix culture.

Theory
The three domains of life are Bacteria, Archaea, and Eukarya (Fig. 6.1). Bacteria, along with
actinomycetes and cyanobacteria (blue-green algae) belong to the prokaryotes while eukaryotes or
eukarya include Fungi, Protozoa, Algae, Plant, and Animal cells. Viruses are obligate intracellular
parasites that belong to neither of these two groups.

Figure 6.1. Three of life from Rising and Reysenbach (2002) and Woese (1987).

Activated sludge is a type of secondary treatment whose primary role is to remove most of the
dissolved solids remaining in the waste stream after primary treatment. Activated sludge is an
enrichment culture of micro and macro-organisms that remove (or change) components considered
to be pollutants. In the activated sludge process, microorganisms are mixed with wastewater. The
microorganisms come in contact with the biodegradable materials in the wastewater and consume
them as food. The microorganisms settle out as sludge when wastewater is in settling tank.

There are five major groups of microorganisms generally found in the aeration basin of the activated
sludge process:

Bacteria-Aerobic bacteria remove organic nutrients

For designing and processing an efficient activated sludge system, it is important to know
7
microbiota of the sludge. In nature, one of the most important function of bacteria is decomposing
organic matters sourced from other living organisms. Bacteria are also the most important members
of an activated sludge process. They form the first level of trophic levels in activated sludge. Aerobic
and facultative bacteria are used for energy production in many organic waste reactor or aeration
tank.
Generally activated sludge includes bacteria belonging to genus Pseudomonas, Zooglena,
Achromobacter, Flavobacterium, Nocordia, Bdellovibrio, Mycobacterium.
Nitrosomonas and Nitrobacter species responsible from nitrification are also present in the sludge.
Sphaerotilus, Beggiotoa, Thiothrix, Lecicothrix and Geotrichum are flamentous community of
activated sludge. As bacteria consumes organics during treatment process, the other bacteria also
gain some important functions, such as Rotifers consumes small biological flocculated particles and
protozoa consumes flocculated bacteria. Flocs may range between 1 m to even more than 1 mm
(Şahinkaya, 2011; Mara and Horan, 2003).

Protozoa-Remove & digests dispersed bacteria and suspended particles. They keep bacteria
population under control. Main types are below:

-Amoebae: Little effect on treatment, they die off as amount of food decreases

-Flagellates: Feed primarily on soluble organic nutrients

-Ciliates: they have different functions. They feed on both bacteria and nutrients, help treatment
process.

Metazoa-Multicellular organisms larger than protozoa. They have no role in nutrient removal.

Filamentous bacteria-bulking sludge (poor settling & turbid effluent)

Algae and Fungi-Fungi is present with pH changes & older sludge.

The microbiological evaluation of the activated sludge systems is directly related to the operation
of the system and providing the desired efficiency parameters. When the system is operating under
ideal operating conditions, there is no microbiological problem in the system. In the case of normal
operation of the system (ie. the pH of the wastewater coming into the treatment system = 7.0,
temperature t = 20 0C, and N, C, P, the ratios are appropriate), bacteria are the dominant
microorganism type of the activated sludge systems.
The substrate type and concentration in the wastewater affects the species distribution of bacteria in
activated sludge systems. If the wastewater coming into the treatment system is rich in protein;
Alcaligenes sp., Flavobacterium sp. and Bacillus sp. are the dominant bacterial species. If the
wastewater from the treatment system is rich in carbohydrates and
hydrocarbons, Pseudomanas sp. increases in number in the activated sludge system; Even in the
absence of oxygen or in very low oxygen levels, Pseudomonas can continue its activities.
After Bacteria, Protozoas are the most important group of microorganisms in activated sludge
systems when operated under normal conditions. Protozoa feed on bacteria. The types of Protozoa
in activated sludge systems depend on the type and amount of substrate in the environment. If the
ratio of the substrate amount to the microorganism mass is high, the motile free-circulating Ciliate
species dominate the medium. These species are important members in activated sludge. The most
8
important among them; Trachelopyhlum pusillum, Vorticella convellaria, Opercularia coarctata.
As the activated sludge system operates steadily and regularly, the Protozoa species in the system
remain stable. Under normal conditions, Rotifers are also important microorganisms in the sludge.
Since Rotifers are absolute aerobic microorganisms, there is no ventilation problem in the ponds
where Rotifers are seen. In other words, there is a sufficient amount of oxygen in these sludge ponds.
Algae are not present under normal operating conditions in activated sludge systems. Fungi are
negligible quantity besides other species. pH of the activated sludge affects presence of Fungi, if the
pH is 7.0 or above (alkali) Fungi cannot dominate environment. They like asidic pH values. The
most important type of Fungi seen in case of dominance in the active sludge system is Fisarium sp.
Fisarium sp. grows as filaments. Filamentous microorganisms have poor precipitation properties.
This situation causes precipitation problem of sludge flocks in sedimentation ponds which affects
quality of treatment (Şahinkaya, 2011; Mara and Horan, 2003).

Practice 1.
Isolation of aerobic heterotrophic bacteria from activated sludge sample.
Procedure:
-Dilute wastewater sample provided for each group as 10-4 and 10-6 dilutions.
-Take 200 ml from each dilution and spread on nutrient agar surface.
-Incubate for 24 h at 30°C.
-Examine the colonies in next laboratory session.

Practice 2.
Isolation of bacteria colonies from soil sample.
The purpose of this lab is to calculate the CFU/mL and describe each of the macro morphologies
observed from soil samples. Various bacterial communities will be observed either in the numbers
of viable colonies supported, the types of macro morphologies isolated, or both.

Procedure:

-Dig the Earth surface 10 to 12 cm deep and collect soil sample bellow 12 cm.
-Take test tubes and take distilled water in test tubes and label each tube.
-Take 1 gram of soil sample from the collected soil sample and make a solution in the test tubes
which having 0.9% sterile NaCl saline solution.
-Shake the tube vigorously to separate the bacteria from the soil particles.
-With a micropipette, transfer 500µL to a sterile micro centrifuge tube. If the solution is too
concentrated and soil is getting stuck in the tip, add more sterile saline in 10mL increments until the
problem ceases. Record in your lab-notebook the final volume of saline in which you re-suspended
the 1g of soil.
-Close the cap of the microfuge tube and vortex for at least 30 seconds to thoroughly mix the
bacterial cells into the 500µl of sterile saline.

9
-Label 3 sterile micro centrifuge tubes containing 900µL of 0.9% sterile saline with the dilution
factors: “10-1, 10-2, 10-3”.
-Using a micropipette and a sterile tip, transfer 100μL of cells from your undiluted (100) tube
containing 500µL of re-suspended soil to the tube labeled 10-1 dilution. Vortex for at least 30
seconds to thoroughly mix the cells into the saline.
-Continue with 1:10 serial dilutions (as shown below).

-For the purring the sample in the media plates Spread Method is used.

-Plate 100µL (0.1 mL) of each of the 100, 10-1, 10-2, 10-3 dilutions on the appropriate agar plates.
Incubate the plates at 30°C for 24-48 hours.

Practice 3.

Microscopic examination of wastewater procedure

-Take 50 μL of sample, place on the slide and cover with cover slip, examine under microscope and
draw the microorganisms seen under 100X objective.
-Count the bacterial cells and protozoa cells under objective area

Materials
5 ml wastewater for each group, Soil sample, Sterile falcon tubes, 0.9% NaCl saline solution, NA
Medium, Glass rod, Syringe, Beaker, Flask, Graduated cylinder, Burner, Ethanol, Slides and cover
slips, Micropipettes

10
Result

Calculation of CFU/mL

-Pick the plate with 30–300 colonies and write down the total number of colonies of that plate
in your lab notebook.
-Calculate the original colony forming units per mL (CFU/mL) using the following formula:

𝐶𝐹𝑈 # 𝑜𝑓 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠
= × 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
𝑚𝐿 𝑚𝐿 𝑝𝑙𝑎𝑡𝑒𝑑

Table 1. Colony Counts from Nutrient Agar Plates

Date Plated Dilution Factor Total # Colonies CFU/mL

… … … …
Discussion
Record in your lab notebook different descriptions and total number of colonies that you
cultured. Include: colony ID, date plated, size, shape, color, texture, and total number of similar
colonies you cultured. Use only the standard descriptions (shown in below Figure).

11
EXPERIMENT 7: DISINFECTION
THE CONTROL OF MICROORGANISMS BY CHEMICAL AND
PHYSICAL FACTORS

Objective: be able to perform antibiotic sensitivity test on a bacterial culture

Theory:
The practices of this unit are concerned with two aspects of microbial growth: promotion and
control. On one hand, the microbiologist is concerned with providing optimum growth
conditions for maximization of growth. The physician, nurse and other members of the medical
arts profession, on the other hand, are concerned with the limitation of microbial populations
in disease prevention and treatment. For supplying safe drinking water, and also for the safe of
water used for other purposes (such as ponds or swimming pools) disinfection has to be applied
to inhibit the growth of pathogenic microorganisms. The final step of wastewater treatment is
disinfection. Mostly chlorination is used for disinfection of drinking water. Although
ozonization is an expensive one, it is one of the widely used disinfection method especially for
swimming pools.
Microbial control by chemical and physical means involves the use of antiseptics, disinfectants,
antibiotics, ultraviolet light, and many other agents.
In this section, practices representing both physical and chemical methods of control will be
presented.

THE ANTIBIOTIC - SENSITIVITY TESTING

Specific Objectives:
I. be able to perform an antibiotic-sensitivity test on a bacterial culture.
2. be able to distinguish the relative resistance and sensitivity of bacterial cultures to selected
antibiotics.

Antibiotics (anti; against, bios; life) form an interesting and therapeutically useful group of
compounds. These chemicals are characterized as being metabolic by products of one
microorganism that is able to kill other microorganisms (bactericidal). The production of
antibiotics by microorganisms is a common phenomenon. One of the first reported discoveries
was made by bacteriologist Alexander Fleming the crude antimicrobial extract be obtained from
Penicillium notatum was named penicillin.
A vast array of antibiotics has been isolated, synthesized, and subsequently used in the treatment
of diseases caused by a variety of pathogenic microorganisms. It is important to note that the
sensitivity of microorganisms to an antibiotic varies. Therefore, the selection of an appropriate
chemotherapeutic agent is extremely important.
The antimicrobial activity of an antibiotic may be tested by several methods designed to
determine the smallest amount of the agent needed to inhibit the growth of a microorganism.

12
The resulting value is known as the minimal inhibitory concentration (MIC). This exercise will
demonstrate the features of the commonly used agar-diffusion method.
In the basic agar-diffusion procedure, a petri plate containing a suitable medium is heavily
inoculated with the microorganism whose antibiotic sensitivity is to be determined. Filter-paper
disks, each containing defined concentrations of a specific antibiotic, are removed from
individual containers and placed onto the agar surface.
The relative effectiveness of different antibiotics provides the basis for a sensitivity spectrum
of the organism. The information, together with various pharmacological considerations, is
used in the selection of an antibiotic for treatment.

Practice 1
Effect of disinfectant on bacterial growth: Place 0.2 mL of E. coli culture on NA-plates provided
and spread these on the agar surface so as to obtain a lawn (dense) culture. Use a sterile (dipped
into alcohol and flamed) spreader to do this. Put 1 mL of concentrated disinfectant (Zephiran
(benzalkonium chloride 10%)) into a test tube containing 9 mL sterile water using a sterile pipette
and mix thoroughly. Transfer 1 mL from 1/10 dilution into another 9 mL sterile distilled water to
obtain 1/100 dilution. Carry on two more times like this to obtain 1/1000 and 1/10000 dilutions
respectively. Then soak two paper disks in each dilution tube. Transfer these disks onto the lawn
(dense) cultures prepared and mark the proper dilutions on the lid of petri dishes. Place petri dishes
upside-down in the 35 oC incubator. Check growth after 24-48 h. Note the maximum dilution
giving no growth under and around disinfectant-disks. Measure inhibition zone around each disk.
Each disc will absorb 30 ul of solution. Calculate how much disinfectant was absorbed by each
disk. Do this calculation for each concentration of antibiotic and disinfectants.

Practice 2
Effect of antibiotics on bacterial growth: Prepare 300 ug/mL antibiotic solution and prepare 10
and 100 times diluted antibiotic solution from this stock. Soak paper disks into the antibiotic
solution with different concentrations. Place antibiotic-impregnated (absorbed) discs on one of the
inoculated plates prepared as described in Practice 1. The antibiotic concentration which each
paper disc is impregnated with should be indicated on the agar plate where the disks are located.
Incubate plates at 35 oC. Observe any growth after 24-48 h. Measure inhibition zone around each
disk.

Practice 3
Effect of chlorine on bacterial growth
Repeat Practice 1 using sodium hypochlorite instead of zephiran (benzalkonium chloride 10%).

13
EXPERIMENT 8: NITRIFICATION AND DENITRIFICATION

Objective: To monitor important microbial transformations of inorganic nitrogen compounds

in sample.
• Add nitrate to tube and measure semi-quantitatively after one week incubation
• Re-measure it for nitrate to determine whether nitrification and/or denitrification have
occurred

Theory:
Nitrate is a negatively charged ion, which is not absorbed to cation exchange sites (of soil)
which are also negatively charged. In contrast, the positively charged ammonium ion is
retained in the soil. Because nitrate is easily washed away from the soil, it will tend to
accumulate in the water environment. Nitrogen in the form of nitrate leads to reduced plant
uptake of nitrogen and lower nitrogen use efficiency. It can also result in contamination of
groundwater. Intake of potable water high in nitrates can result in methemoglobinemia.
However, nitrification can be a beneficial reaction during wastewater treatment since it allows
for subsequent denitrification, thereby reducing the nitrate content of effluent.
Nitrification and denitrification are both essential transformations within the nitrogen cycle.
Nitrification involves the oxidation of a reduced form of nitrogen, commonly ammonium, to
nitrate in a two-step reaction. Typical nitrifying organisms for the first step belong to the genus
Nitrosomonas, which convert ammonium to nitrite:
NH4 + 3/2 O2 NO2- + 2H+ + H2O + Energy

The second step in nitrification concerns the oxidation of nitrite to nitrate, a reaction
commonly carried out by Nitrobacter sp.:
NO2-+3/2O2 NO3- + energy

Both Nitrosomonas and Nitrobacter are chemoautotrophic organisms which use the energy
derived from the oxidation of ammonium or nitrite to fix carbon dioxide. Because so much
less energy is released from the second step than the first (ca. 100 moles of nitrite must be
reduced to fix the same amount of CO2 as 35 moles in the first step), very little nitrite
accumulates in the soil (Atlas and Bartha, 1987).
Control of the nitrification is important in agricultural systems as the negatively charged
nitrate is not retained by positively charged soil particles.
Normally, denitrifiers are facultative anaerobes which prefer to use oxygen as the terminal
electron acceptor in respiration if it is available. However, when oxygen becomes limiting, they
can use nitrate as the terminal electron acceptor. Dinitrogen gas is one of the common end

14
products of denitrification. However, the greenhouse gas N2O is also produced. This
contributes to global warming (Pepper et al., 1996).
One major heterotrophic organism involved in denitrification is Pseudomonas denitrificans:
C6H12O6 +4NO3- 6CO2 + 6H2O+ 2N2 + energy

Here, the degree to which denitrification takes place is limited by the amount of oxidizable
carbon present in the system.
An important autotrophic organism is Thiobacillus denitrificans:
2NO3- + S + H2O +CaCO3  CaSO4 +N2+ energy

Some microorganisms can use nitrite and nitrate as nitrogen source for growth and they
reduce the nitrogen to ammonium form, which has key role in amino acid synthesis. The
reaction of denitrification can be observed by adding a little amount of KNO3 to the Nutrient
Broth. When microorganisms are inoculated into the medium, they grow at the bottom of the
broth and if they have the enzymes specific for denitrification, the final product KNO 2 is
produced. KNO2 is accumulated in the medium. Sulfanilic acid and α–naftol is used for
chemical determination of the nitrite salt. Nitrites can be converted into NH 3 based on the
enzyme types of the microorganisms have:
+4𝐻
→
2𝐾𝑁𝑂2 → 2𝑁𝐻2 𝑂𝐻 → 2𝑁𝐻3
The produced NH3 is identified using Nessler solution:

2𝐻𝑔𝐼42− + 2𝑁𝐻3 ⇆ 𝑁𝐻2 𝐻𝑔2 𝐼 3 + 𝑁𝐻4+ + 5𝐼 − (Orange color)

The produced NH3 can be degraded up to nitrogen gas:

2𝑁𝐻𝑂2 → 2𝑁𝑂 → 𝑁2 𝑂 → 𝑁2
−𝐻2 𝑂 −𝐻2 𝑂 −𝐻2 𝑂

The produced nitrogen gas is identified by observing the gas accumulation in Durham tubes.

Experiment
Material:
1) Pseudomonas aeruginosa, B. subtilis, E. coli (46 houred agar slants)
2) Nitrite Broth
Add 1g KNO2, 0.5 g NaCl, 2g Peptone into distilled water. Bring volume to 1.0 L with
remaining water.
3) Nitrate Broth
Add 1g KNO3, 0.5 g NaCl, 2g Peptone into distilled water. Bring volume to 1.0 L with
remaining water.
15
 4) Test tubes
5) Durham tubes
6) Sulfanilic solution
7) N, N-Dimethyl-1-naphthylamine solution
8) Nessler solution
9) Zinc dust
10) Sludge sample

Procedure:
1. 10 tubes containing nitrite and nitrate broth are prepared.
2. P. denitrificans, E. coli, B. subtilis and activated sludge are inoculated into four different tube
that contains nitrite broth and nitrate broth.
3. Tubes 1 are control samples containing only media. Pseudomonas is cultivated into second
tubes. Bacillus is cultivated into third tubes. E. coli is cultivated into fourth tubes. Into fifth
tubes, activated sludge is added.
4. All tubes are incubated at 30ºC.
5. Two of each of these samples are prepared. 10 tubes are incubated for 1 day. The other 10
tubes are incubated for 7 days.
6. Experiments start with 1 day incubated tubes.
7. From the start of the 1st day, 1 ml of inoculum is transferred using sterile pipettes. 3 drops
of sulfanilic acid solution and 2 drops of N, N-Dimethyl-1-naphthylamine solution is added
onto each tube. If there is nitrite in the medium, compound colored pink-red is occurred. This
color indicates the (+) results for nitrites.
8. After a (+) result for nitrites, one more day incubation is done to ensure the NH3 production.
After that, 1ml cultures are transferred from the test tubes and 1-2 drop Nessler solution is
used to identify the NH3. The yellowish-orange color indicates the NH3 in the medium.
9. Any N2 gas can easily be detected by putting the Durham tubes inversely into the Nitrate
Broth.
10. Zinc can be used for determination of nitrate reduction to ammonia:
If there is no gas and no color change, nitrate has been not reduced. In this event, remove 2
ml from the tube and place into a second test tube. Add a pinch of zinc dust to the tube, rotate
to mix, and allow the tube to stand for 10 minutes. The zinc reduces nitrate to nitrite. If a red
color appears in the bottom of the tube, it may be assumed that nitrate was not reduced. If
no red color appears, nitrate reduction has occurred, but the resulting nitrite has become
further reduced to ammonia.

References:
https://www.epa.gov/sites/production/files/2015-12/documents/9131.pdf METHOD 9131
TOTAL COLIFORM: MULTIPLE TUBE FERMENTATION TECHNIQUE
https://microbeonline.com/probable-number-mpn-test-principle-procedure-results/ Most
Probable Number (MPN) Test: Principle, Procedure and Results
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17