BML 2212 Food Air and Water Microbiology CAT 11

BENJAMIN MUTISYA

BMLSUP/2023/39391

1. Describe presumptive and confirmatory coliform tests in food and

water microbiology and their results interpretation 15mks

The presumptive and confirmed tests are one of the components of Most
Probable Number (MPN) tests, which is a statistical method, used for estimating
the concentration of viable microorganisms such coliform bacteria in a given
sample.

It is commonly used in estimating microbial populations in soils, waters, and

agricultural products. MPN test is particularly useful with samples that contain
particulate material that interferes with plate count enumeration methods.

1) The presumptive test

The presumptive test is a preliminary screening method used to detect the

presence of coliform organisms in water samples. It involves utilizing a series
of fermentation tubes containing lactose broth with known concentrations.

Purpose of the test

The purpose of this test is to quickly assess whether the water source or food
source may be contaminated with coliform bacteria, which can indicate
potential fecal contamination and the presence of harmful pathogens.

Principle of the test

Water or food sample to be tested is diluted serially and inoculated in lactose
broth, coliforms if present in the sample utilizes the lactose present in the
medium to produce acid and gas. The presence of acid is indicated by the color
change of the medium(red to yellow) and the presence of gas is detected as gas
bubbles collected in the inverted Durham tube present in the medium. The
number of total coliforms is determined by counting the number of tubes giving
positive reaction (i.e both color change and gas production) and comparing the
pattern of positive results (the number of tubes showing growth at each
dilution) with standard statistical tables.

Materials required

1. Sterile test tubes or bottles with lactose-containing broth (e.g., lactose

broth or MacConkey broth)
2. Durham tubes or gas detection vials
3. Sample to be tested (food or water)
4. Incubator set to the appropriate temperature (usually 35-37°C)
5. Inoculating loop or pipette
6. Gas production chart or statistical tables for MPN estimation (if using
MPN method)
7. Personal protective equipment

Preparation of the media

Prepare MacConkey broth or lactose broth, in two concentrations: single

strength and double strength.

For samples of untreated or polluted water:

Dispense the double strength medium into 10 tubes, with each tube containing
10mL. Similarly, dispense the single strength medium into 5 tubes, with each
tube holding 10 mL. Ensure that a Durham tube is placed in an inverted position
within each test tube.

For samples of treated water:

Dispense the double strength medium into 5 tubes, each containing 10mL.
Additionally, pour 50 mL of single strength medium into a separate bottle. Place
a Durham tube in an inverted position within each container.

Examine the tubes to ensure that the Durham tubes are completely filled with
the liquid medium, with no air bubbles present.

Finally, sterilize all the prepared tubes and bottle by autoclaving them at 15 lbs
pressure (121°C) for a duration of 15 minutes. This step ensures that the
medium is free from any contaminants before the test begins.

Procedure

1. Label the test tubes or bottles with sample identifiers and dilutions
2. If the sample is solid (e.g., food), prepare a homogenized suspension by
blending or grinding a representative portion of the sample with a suitable
diluent (e.g., sterile saline solution or peptone water). For liquid samples
(e.g., water), proceed to the next step.
3. Aseptically transfer 10 mL of the water sample/ diluted food sample
using a sterile pipette into the 5 tubes containing 10 mL of double
strength medium.
4. Add 1 mL of the water sample / diluted food sample to 5 tubes
containing 10 mL of single strength medium. For the remaining 5 tubes
with single strength medium, add 0.1 mL of the water sample.
5. Add a Durham tube or gas detection vial to each inoculated tube or bottle.
The Durham tube should be inverted before insertion to trap any gas
produced during fermentation.
 6. Cap the tubes or bottles tightly to prevent contamination and incubate
them at 35-37°C 24 hours.
If no positive reactions observed, re-incubate the tubes for up to 48 hours.
Results and interpretation

RESULTS DESCRIPTION
POSITIVE Formation of 10% Gas in the Durham tube within 24 to 48 hours
RESULTS Turbidity in the growth medium
Color change of the growth medium from red to yellow indicates
presence of coliform bacteria in the test sample

NEGATIVE No visible growth observed in the growth medium

RESULTS No gas formation in the Durham tube
No colour change in the growth medium
Indicates the absence of coliform bacteria.
Suggests a lower likelihood of fecal pollution.
Note: Does not guarantee absence of other microorganisms.

Results interpretation

The number of total coliforms is determined by counting the number of tubes

giving positive reaction (i.e both color change and gas production) and
comparing the pattern of positive results (the number of tubes showing growth
at each dilution) with standard statistical tables.

Positive Result:

The combined presence of gas, turbidity, and color change in the medium
signifies a positive presumptive test for coliform bacteria. This result suggests
the potential presence of fecal pollution in the water sample.
It’s essential to understand that the test is termed “presumptive” because, under
the conditions set for this test, several other types of bacteria can produce results
similar to coliforms. Therefore, while a positive result indicates the likelihood
of coliform presence, it is not definitive.

Negative results

A negative result suggests that the test sample does not contain coliform
bacteria and is likely free from fecal pollution.

If the presumptive test is negative, no further testing is performed, and the water
source is considered microbiologically safe. If, however, any tube in the series
shows acid and gas, the water is considered unsafe and the confirmed test is
performed on the tube displaying a positive reaction.

2) CONFIRMATORY TEST OF MPN TEST

The Confirmed Test is a crucial step in the Most Probable Number (MPN) Test,
designed to validate the presence of coliform bacteria in water samples. It’s
essential to understand that the gas produced in the Presumptive Test does not
guarantee the presence of coliforms in the water sample. Water contains various
microorganisms, some of which, like certain yeasts and Clostridium species,
can ferment lactose, producing both acid and gas. This can lead to false positive
results in the Presumptive Test. Therefore, the Confirmed Test is vital to
ascertain the presence of coliforms.

PUPOSE OF THE TEST

The primary objective of the Confirmatory MPN Test is to ensure the presence
of coliform bacteria by examining the tubes that showed positive results in the
Presumptive Test.

The Confirmed Test can be conducted in two primary ways:

1. Brilliant Green Lactose Bile Broth (BGLB)

 2. Eosin Methylene Blue Agar Medium (EMB)

1) Brilliant Green Lactose Bile Broth (BGLB)

This method involves using BGLB, a selective medium designed to detect

coliform bacteria in water, dairy products, and other food items. The selective
agent in this medium is lactose. Additionally, the broth tube contains a Durham
tube to capture and detect gas production. The brilliant green dye present in the
BGLB medium inhibits the growth of gram-positive bacteria, ensuring that only
the desired coliform bacteria grow in the medium.

Preparation of BGLB Medium:

Composition

a) Peptone: 10 g
b) Lactose: 10 g
c) Bile salt: 20 g
d) Brilliant green: 0.0133 g
e) Distilled water: 1 L

Sterilize the medium by autoclaving for 15 minutes at a temperature of 121

degrees Celsius.

Procedure

1) Before inoculation, shake the positive presumptive tubes gently to ensure

an even distribution of the bacteria.
2) Using a sterile loop, transfer a loopful of culture from the positive
presumptive tube into the BGLB fermentation tube.
3) After inoculation, incubate at a consistent temperature of 35 degrees
Celsius for 48 hours, allowing sufficient time for the bacteria to grow and
produce observable results.
 Observation:

Examine the presence of gas production in the inverted Durham tube or turbidty
in the growth medium.

RESULTS DESCRIPTION
POSITIVE There is gas production indicated by presence of as bubbles
in the inverted Durham because of the fermentation process
of the coliform bacteria.
Presence of Turbidity(cloudiness or haziness) in the broth,
indicating bacterial growth

NEGATIVE No gas production

No turbidity in the growth media

Result Interpretation:

If gas production or turbidity is observed in the BGLB medium, it indicates the

presence of coliforms in the sample. This gas production is a definitive sign of
coliform bacteria, as they are known to ferment lactose, producing gas in the
process.

2) Eosin Methylene Blue Agar Medium (EMB)

Eosin Methylene Blue (EMB) agar medium is a selective and differential

medium primarily used to isolate and differentiate coliforms from other
bacterial species.

Purpose of the test

The EMB agar medium is used in the microbiological analysis of water
samples. Its selective and differential properties ensure the accurate
identification and differentiation of coliform bacteria from other microbial
species.

Principle of the test

When a sample from a positive tube is streaked on this medium and incubated,
typical coliform bacteria like E. coli and Enterobacter aerogenes exhibit growth
and form distinctive red to black colonies with dark centers or a sheen. In
contrast, other bacteria like Salmonella typhi might show growth but produce
colorless colonies, while some, like S. aureus, might not grow at all.

Preparation of EMB Medium:

components:

i. Peptone: 10 g
ii. Agar: 15 g
iii. Lactose: 10 g
iv. Eosin Y: 0.4 g
v. Methylene blue: 0.065 g
vi. Dipotassium hydrogen phosphate: 2 g
vii. Distilled water: 1L

Sterilize the EMB agar by autoclaving it for 15 minutes at 121 degrees Celsius.

Once sterilized, pour the molten EMB agar onto sterilized Petri plates. Allow
the medium to solidify, creating a firm surface suitable for bacterial growth.

Procedure

i. Prior to inoculation, shake the positive presumptive tubes gently to ensure

a uniform distribution of bacteria.
 ii. Using a sterile loop, streak a loopful of culture from the positive
presumptive tube onto the solidified EMB agar surface.
iii. Place the inoculated Petri plates in an incubator set at 35 degrees Celsius
and allow the plates to incubate for 48 hours, providing adequate time for
bacterial growth and differentiation.

Observation:

After the incubation period, examine the Petri plates for bacterial colonies on
the EMB medium. Typically, three distinct types of colonies can develop:

Typical colony: These colonies are characterized by their nucleated appearance

and may exhibit a metallic sheen. This sheen is indicative of coliform bacteria,
which ferment lactose, producing acid that reacts with the dyes in the medium.

Atypical colonies: These colonies appear non-nucleated, opaque, and mucoid.

They represent bacterial species that do not produce the characteristic sheen.

Negative colonies: These colonies differ in appearance from both typical and
atypical colonies and represent other bacterial species.

Result Interpretation

Results
positive  Formation of gas in the lactose broth medium.
 Presence of colonies with a greenish metallic sheen on Eosin
Methylene Blue agar medium.
 Indicates the presence of coliform bacteria or a member of the
coliform group in the test sample.
 High-temperature presence of typical colonies suggests the presence
of thermotolerant Escherichia coli (E.coli).
Negative  Absence of gas formation in lactose broth medium.
 Failure to demonstrate coliform-like colonies (no greenish metallic
 sheen) on EMB agar medium.
 Indicates the absence of coliform bacteria in the water sample.

Positive result:

The presence of typical colonies with a metallic sheen on the EMB agar is a
clear indication of coliform bacteria. This sheen is a result of the interaction
between the acid produced by lactose fermentation and the dyes in the EMB
medium. This sheen is a hallmark of coliform bacteria and differentiates them
from non-coliform bacteria, which do not produce such a sheen.

Negative results:

A negative result is indicated by the absence of coliform-like colonies on the

EMB agar. Specifically, if there are no colonies exhibiting the characteristic
greenish metallic sheen, it suggests that coliforms are not present in the sample
or they are present in negligible amounts.

2. Explain what constitutes air microbiology and how it is

assessed microbiologically 10mks

Air microbiology is the study of suspended microorganisms in the air. It’s also
known as, aero microbiology. It involves study of microbes and their airborne
spores that are invisible to the naked eyes such bacteria, fungi, viruses and other
microbes. It encompasses the investigation of the types, abundance, distribution,
behavior, and ecological roles of microorganisms found suspended in the
atmosphere.

Air microbiology constitute indoor air microbiology and outdoor air

microbiology

1. Indoor air microbiology refers to the study of microorganisms present in

enclosed or confined indoor spaces. This field of microbiology focuses on
 the assessment and management of microbial contaminants found
indoors, including bacteria, fungi, viruses, and other indoor bioaerosols.
Indoor air microbiology investigates sources of indoor microbial
contamination such as humans, pets, plants, soil, water damage, and
building materials. It examines the transmission routes of indoor airborne
microorganisms, including respiratory droplets, aerosols, and surface-to-
air dispersal.

Relevant areas include; Aeromicrospora of Pharmacy;Aeromicroflora of

Hospitals and Aeromicroflora of Storage Materials (e.g., libraries, wall
paintings)
2. Sources of air microbes

Air microbes originate from various natural and anthropogenic sources. In

the outdoor environment, sources include soil, vegetation, water bodies,
wildlife, decaying organic matter, and atmospheric dust. Indoors, human
occupants, pets, plants, indoor surfaces, HVAC systems, and moisture
contribute to indoor air microbiota. Anthropogenic activities such as
industrial processes, agriculture, waste management, and certain occupations
also release airborne microorganisms. Understanding these diverse sources is
crucial for assessing environmental microbiology, indoor air quality, and
potential health risks associated with airborne microorganisms. Effective
control measures can help mitigate exposure and protect human health and
the environment.

3. Factors affecting air microbes

Various factors influence the presence and behavior of air microbes.

Environmental conditions such as temperature, humidity, sunlight, wind, and
seasonality affect microbial survival, growth, and dispersal. Human
activities, including occupancy, ventilation, hygiene practices, and
occupational exposure, contribute to indoor and outdoor microbial dynamics.
 Additionally, natural sources like vegetation, soil, and water bodies, along
with anthropogenic sources such as industrial processes and transportation,
introduce microorganisms into the air. Understanding these factors is crucial
for assessing air microbiology, predicting microbial dispersion, and
implementing control measures to maintain air quality.

4. Outdoor air microbiology refers to the study of microorganisms present

in the atmosphere and outdoor environments. This branch of
microbiology focuses on the characterization and understanding of
microbial communities, including bacteria, fungi, viruses, and other
microorganisms, that exist in outdoor air. Outdoor air microbiology
examines the sources, distribution, abundance, diversity, and dynamics of
airborne microorganisms in natural and anthropogenic settings.

The constituents of air microbiology include various types of microorganisms

such as bacteria, fungi, viruses, archaea, and microbial fragments. These
microorganisms can be found in different forms, including free-living cells,
spores, pollen grains, and other particulate matter.

i. Bacteria:
Bacteria are diverse microorganisms that can be found virtually everywhere,
including in the air. They play crucial roles in nutrient cycling, decomposition,
and various ecological processes.

Airborne bacteria can include species such as Staphylococcus, Streptococcus,

Bacillus, and Pseudomonas. While some are harmless, others can cause
respiratory infections, foodborne illnesses, or skin infections.

Bacteria can become aerosolized through activities like sneezing, coughing, or

mechanical agitation of surfaces where they reside.Airborne bacteria originate
from various sources such as soil, water bodies, vegetation, human and animal
activities, and industrial processes. They can become airborne through
processes like wind erosion, agricultural activities, and aerosolization of
contaminated water.

ii. Fungi:

Fungi encompass a wide range of organisms, including molds, yeasts, and

mushrooms. They exhibit diverse morphologies and ecological niches, ranging
from decomposers to symbionts and pathogens.

They can release spores into the air, which can remain airborne for extended
periods.Airborne fungi can cause allergies, asthma, and other respiratory
problems in susceptible individuals. Some fungi produce mycotoxins, which
can be harmful to humans and animals if inhaled or ingested.Common airborne
fungi include Aspergillus, Penicillium, Cladosporium, and Alternaria.

iii. Viruses:

Viruses are infectious agents that require a host cell to replicate. Airborne
viruses can be transmitted through respiratory droplets produced when an
infected person coughs, sneezes, or talks.Airborne viruses can cause a range of
diseases, from the common cold and flu to more severe illnesses like COVID-
19 and SARS.

Examples of airborne viruses include influenza viruses, rhinoviruses (cause of

the common cold), coronaviruses, and respiratory syncytial virus (RSV).

iv. Protozoa:

Protozoa are single-celled eukaryotic organisms found in diverse habitats,

including soil, water, and air. While less common in the air compared to
bacteria and fungi, certain types of protozoa can become aerosolized.
Airborne protozoa may include species such as Acanthamoeba and Naegleria.
Inhaling airborne protozoa can potentially lead to respiratory infections or other
health issues.

v. Algae:

Algae are photosynthetic organisms found mainly in aquatic environments, but

some species can release spores or fragments that become airborne.

While airborne algae are less common compared to bacteria and fungi, they can
contribute to the microbial diversity of the air, particularly in coastal or aquatic
environments where algal blooms occur.

vi. Dust and Particles:

Airborne dust and particles can serve as carriers for various microorganisms,
including bacteria, fungi, and viruses. These particles can originate from
sources such as soil, pollen, pollutants, and organic matter.Microorganisms
attached to dust particles can be transported over long distances in the air,
affecting air quality indoors and outdoors.Certain particles, such as those from
construction activities or indoor renovations, can contain higher concentrations
of microorganisms, posing potential health risks to occupants.

vii. Endotoxins and Exotoxins:

Endotoxins are lipopolysaccharides found in the outer membrane of certain

Gram-negative bacteria. When these bacteria die, endotoxins are released and
can become aerosolized.

Exotoxins are protein toxins secreted by various bacteria, including those that
cause diseases like tetanus, diphtheria, and botulism. These toxins can also
become airborne and pose health risks if inhaled.

viii. Bioaerosols:
Bioaerosols refer to airborne particles of biological origin, including
microorganisms, their fragments, and byproducts. These particles can be
generated from natural sources (e.g., soil, vegetation) or human activities (e.g.,
agricultural practices, indoor air pollution).Bioaerosols can contribute to
respiratory allergies, infections, and other health issues, particularly in indoor
environments with poor ventilation or high levels of microbial contamination.

ix. Allergens and Toxins:

Allergens: Airborne allergens are substances that can trigger allergic reactions
in susceptible individuals, leading to symptoms such as sneezing, coughing,
wheezing, and skin rashes. Common airborne allergens include pollen grains,
fungal spores, dust mites, animal dander, and certain proteins produced by
bacteria and fungi.

How these air microbiology constituents are assessed microbiologically

Assessing the constituents of air microbiology involves various methods to

evaluate the presence and abundance of microorganisms in the air. These
microbiological assessment techniques include;

1) Air sampling method

Air sampling involves collecting air samples from the environment using
specialized equipment. There are various methods for air sampling, including:

 Impaction: Air is impacted onto a solid agar surface, capturing

microorganisms present in the air onto the agar medium. The agar plates
are then incubated to allow the growth of colonies, which can be counted
and identified.
 Impingement: Air is drawn through a liquid medium, trapping
microorganisms in the liquid. The liquid is then plated onto agar or
analyzed directly for microbial content.
  Filtration: Air is passed through a filter that traps microorganisms. The
filter is then transferred to a growth medium or analyzed directly for
microbial content.
2) Microbial Culture:

After air sampling, microbial cultures are prepared by streaking or inoculating

the collected samples onto appropriate growth media. These media may be
selective, allowing specific types of microorganisms to grow, or non-selective,
allowing a wide range of microorganisms to grow. After incubation under
suitable conditions, colonies can be counted and identified based on their
morphology, biochemical characteristics, and, in some cases, molecular
methods such as PCR or sequencing.

3) Microscopic Examination:

Air samples can be examined under a microscope to directly observe and

identify microorganisms present. Staining techniques such as Gram staining or
fluorescent staining may be used to enhance visualization and aid in
identification.

4) Molecular Techniques:

Molecular methods such as Polymerase Chain Reaction (PCR), quantitative

PCR (qPCR), and DNA sequencing can be used to detect and identify
microorganisms present in air samples. These techniques offer high sensitivity
and specificity and can detect a wide range of microorganisms, including those
that may not readily grow in culture.

5) Biochemical Analysis:

Various biochemical tests may be performed on isolated colonies to identify

specific microorganisms based on their metabolic properties. These tests include
catalase tests, oxidase tests, fermentation tests, and more.
 6) Bioaerosol Samplers:

Specialized samplers are designed specifically for collecting bioaerosols

(airborne particles containing living organisms) from the environment. These
samplers often use impaction, impingement, or filtration methods and may
include additional features such as size-selective sampling to target specific
particle sizes.

7) Total Viable Count (TVC):

In air microbiology, the total viable count (TVC) is a method used to assess the
overall microbial load present in a volume of air. It provides valuable
information about the concentration of viable microorganisms suspended in the
air, which can be indicative of indoor air quality, environmental contamination,
or potential health risks.

8) Flow Cytometry:

In air microbiology, flow cytometry can be utilized to enumerate and

characterize microbial cells present in air samples. This method provides rapid
analysis and can differentiate between various types of microorganisms based
on their size, shape, and fluorescence properties.

9) Fluorescent Particle Counters:

Detects airborne particles based on their fluorescence properties.
Useful for real-time monitoring of particle concentration.

The choice of method depends on factors such as the environment, research

goals, and available resources. Each method has its advantages and limitations,
and researchers often combine multiple techniques for a comprehensive
assessment of air microbiology.