Microbial Growth

1. Determine the decimal reduction time or D-value from the following data. (2020, 1.25
marks)
Table
2. A bacterial cell divides by binary fission 🡪 What does it mean? Describe the events that
take place during bacterial cell division. (2019, 3.5 marks)
Ans: See 5.1 of Brock's book.
3. ❝The population of bacterial cells divides at a constant rate❞ - Elucidate the reasons by
comparing different phases of growth. (2019, 4 marks)
4. What do you mean by exponential growth? Prove that, n = 3.3 (log10N - log10N0). Where,
n = generation time, N0 = initial bacterial number, N= number of bacteria after a given
time. (2018, 2 marks)
Ans: Exponential Growth: A pattern of population increase, where the number of cells doubles
during a constant time interval, is called exponential growth. When the cell number from such an
experiment is graphed on arithmetic (linar) coordinates as a function of time, one obtains a curve
with continuously increasing slope.

5. What are the possible reasons and bacterial responses in stationary phase? (2018, 2
marks)
Ans: Typically, either one or both of two situations limit growth:
(1) an essential nutrient of the culture medium is used up, or
(2) a waste product of the organism accumulates in the medium and inhibits growth. Either way,
exponential growth ceases and the population reaches the stationary phase.
In the stationary phase, there is no net increase or decrease in cell number and thus the growth rate
of the population is zero. Although the population may not grow during the stationary phase, many
cell functions can continue, including energy metabolism and biosynthetic processes. Some cells
may even divide during the stationary phase but no net increase in cell number occurs. This is
because some cells in the population grow, whereas others die, the two processes balancing each
other out. This is a phenomenon called cryptic growth.
6. Differentiate between batch, fed batch and continuous bacterial culture. (2018, 2 marks)
Ans:
7. Briefly describe the practical importance of bacterial growth curve. (2018, 1.5 marks)
Ans: When a bacterial population is cultured , the population size of the bacteria increases showing
a classical pattern. When plotted on a graph, a distinct curve is obtained referred to as the bacterial
growth curve. The growth curve shows four distinct phases : lag phase, log phage, stationary phase
and death phase. The bacterial growth curve can be represented as following figure :

The importance of the Bacterial Growth Curve is widespread.

The study of bacterial growth curves is important when aiming to utilize or inoculate known
numbers of the bacterial isolate, for example to enhance plant growth, increase biodegradation of
toxic organics, or produce antibiotics or other natural products at an industrial scale.
Knowledge of bacterial growth kinetics and bacterial numbers in a culture medium is important
from both a research and commercial point of view.
Growth kinetics is also useful for assessing whether particular strains of bacteria are adapted to
metabolize certain substrates, such as industrial waste or oil pollution.
Bacteria that are genetically engineered to clean up oil spills, for example, can be grown in the
presence of complex hydrocarbons to ensure that their growth would not be repressed by the toxic
effects of oil.
Similarly, the slope and shape of growth curves produced from bacteria grown with mixtures of
industrial waste products can inform scientists whether the bacteria can metabolize the particular
substance, and how many potential energy sources for the bacteria can be found in the waste
mixture.
8. Write down the advantages and limitations of direct microscopic count and viable cell
count. (2018, 1.5 marks)
Ans: Advantages of Direct Microscopic Count
1.Rapid, Simple and easy method requiring minimum equipment.
2.Morphology of the bacteria can be observed as they counted.
3.Very dense suspensions can be counted if they are diluted appropriately.
Microscopic counting is a quick and easy way of estimating
microbial cell number. However, it has several limitations:
1. Without special staining techniques , dead cells cannot be distinguished from live cells.
2. Small cells are difficult to see under the microscope, and some cells are inevitably missed.
3. Precision is difficult to achieve.
4. A phase-contrast microscope is required if the sample is not stained.
5. Cell suspensions of low density (less than about 106 cells/milliliter) have few if any bacteria in
the microscope field unless a sample is first concentrated and resuspended in a small volume.
6. Motile cells must be immobilized before counting.
7. Debris in the sample may be mistaken for microbial cells.

The advantages that have made viable plate counts an important counting process are:
1. The viable plate count is a popular method for determining cell number. The technique is
sensitive and has the advantage of only counting living bacteria, which is often the important issue.
2. Any concentration of microorganism can be easily counted, if the appropriate dilution is plated.
It is even possible to concentrate a solution before counting, as is often done in water analysis,
where bacterial populations are usually at low density.
3. The equipment necessary for performing viable plate counts is readily available in any
microbiology lab and is cheap in comparison to other methods.
4. Finally, by using a selective medium it is possible to determine the number of bacteria of a
certain class, even in mixed populations. These advantages have made viable plate counts a
favorite of food, medical, aquatic and research laboratories for the routine determination of cell
number.
Despite these advantages, viable plate count has some limitations too.
1. One major limitations of the viable plate count is the assumption that each colony arises from
one cell. In species where cells grow together in clusters, a gross underestimation of the true
population results.
2. Great care must also be taking during dilution and plating to avoid errors. Even one error in
dilution can have large effects on the final numbers.
3. If too short an incubation time is used, some colonies may be missed.
4. The temperature of incubation and medium conditions must also be optimized to achieve the
largest colonies possible so that they are easily counted.
5. Finally, this technique takes longer time to determine cell (CFU) numbers.

9. Short notes: ‘Synchronous growth of bacteria.’ (2018, 1.5 marks)

Ans: A synchronous or synchronized culture is a microbiological culture or a cell culture which
contains the cells that are all in the same growth stage. The Synchronous culture also known as the
synchronous growth. A population can be synchronized by manipulating their physical
environments or physical composition of the medium.
If we keep an organism under unfavourable conditions there they will metabolize very slowly, but
will not divide. If we keep the organisms under favourable conditions, then all cells undergo
division and stay at the same phase.The cells of the synchronously growing culture divide at a
time, their growth curve forms a Zig Zag pattern.The easiest way to synchronize bacterial growth
is to add some cytostatic agents so that cells don’t divide and they all maintain the same state of
metabolism and cell cycle. When the cytostatic agent is removed, all cells start to divide at the
same time.Synchronous culture/Synchronous growth of bacteria consists of two phases such as
stationary phase and exponential phase. Synchronous culture helps in the separation of the smallest
cells from an exponentially growing culture. In the laboratory it is used to study the cell cycle. It
is also mportant in the study of genetics and metabolism.
Microbial Growth Control
1. Why is moist heat more effective than dry heat to control microbes? (2020, 2.5 marks)
Ans: Moist heat has better penetrating power than dry heat and, at a given temperature, produces
a faster reduction in the number of living organisms. (Brock 757)
2. Why is sterilization by radiation is a suitable method in food industry? (2020, 1.25 marks)
Ans: Certain foods and food products are also routinely irradiated to ensure sterilization,
pasteurization, or insect deinfestation. Radiation is approved by the World Health Organization
and can be used in the United States for decontamination of foods particularly susceptible to
microbial contamination such as fresh produce, meat products, chicken, and spices. The use of
radiation for these purposes is an established and accepted technology in many countries.
However, the practice has not been readily accepted in some countries such as the United States
because of fears of possible radioactive contamination, alteration in nutritional value, production
of toxic or carcinogenic products, and perceived “off” tastes in irradiated food. (Brock 760)
3. What is HEPA filter and where is it commonly used? (2020, 1.5 marks)
Ans: Depth filters are important for biosafety applications. For example, manipulations of cell
cultures, microbial cultures, and growth media require that contamination of both the operator and
the experimental materials are minimized. These operations can be efficiently performed in a
biological safety cabinet with airflow, both in and out of the cabinet, directed through a depth filter
called a HEPA filter, or high-efficiency particulate air filter.
Control of airborne particulate materials with HEPA filters allows the construction of “clean
rooms” and isolation rooms for quarantine, as well as specialized biological safety laboratories.
HEPA filters typically remove 0.3-µm test particles with an efficiency of at least 99.97%; they
remove both small and large particles, including most microorganisms, from the airstream. (Brock
761)
4. Mention one application of nucleophore filters. (2020, 1 marks)
Ans: Nucleopore filters are commonly used to isolate specimens for scanning electron microscopy.
(Brock 761)
5. Distinguish between sterilization and disinfection. (2019, 1 marks)
Ans: Sterilization is the killing or removal of all viable organisms from a growth medium or
surface. In certain circumstances, however, sterility is not attainable or practical, as in fresh foods.
Microorganisms can be effectively controlled by limiting or inhibiting their growth.
 Disinfection, in contrast, directly targets pathogens, although it may not eliminate all
microorganisms. Specialized chemical or physical agents called disinfectants can kill
microorganisms or inhibit microbial growth. (Brock 756)
6. Why is it necessary to control the growth of bacteria in food while most of them are
beneficial to mankind? (2019, 2.5 marks)
Ans: By controlling the environment and conditions, even if potentially harmful bacteria are
present in the unprepared or raw food, they will not be able to survive, grow, and multiply, causing
illness. (Google)
7. What are the roles of high pressure and radiation in controlling bacterial cells? (2019, 2
marks)
Ans: High Pressure: High-pressure processing (HPP) can improve food safety by destroying the
microorganisms that cause food-borne illness and spoilage that cause diseases. It also leads to the
reduction of fungal toxic metabolites in food products when combined with moderate
temperature. (Google)
Radiation: Several radiation sources are useful for sterilization. Common sources of ionizing
radiation include cathode ray tubes that generate electron beams, X-ray machines, and radioactive
nuclides 60Co and 137Cs, which are relatively inexpensive by-products of nuclear fission. These
sources produce electrons, X-rays, or γ-rays, respectively, all of which have sufficient energy to
efficiently kill microorganisms. In addition, X-rays and γ-rays penetrate solids and liquids, making
them ideal for treatment of bulk items such as ground beef or cereal grains. Radiation is currently
used for sterilization and decontamination in the medical supplies and food industries. (Brock 760)
8. Explain mechanism of action of quaternary ammonium compounds in controlling
microbes. (2019, 2 marks)
Ans: Interact with phospholipids of cytoplasmic membrane. (Brock 765 table)
9. What do you understand by sterilization and commercial sterilization? (2018, 1 marks)

Ans:
10. Differentiate between moist heat and dry heat sterilization. (2018, 2 marks)
Ans:
11. All antiseptics are disinfectants but all disinfectants are not antiseptics. – Explain. (2018,
1.5 marks)
Ans: Disinfectants are chemicals that kill microorganisms, but not necessarily endospores, and are
used on inanimate objects. For example, disinfectants such as ethanol and cationic detergents are
used to disinfect floors, tables, bench tops, walls, and so on.
Antiseptics and germicides are chemical agents that kill or inhibit growth of microorganisms and
that are nontoxic enough to be applied to living tissues. Most of the compounds in this category
are used for handwashing or for treating surface wounds.
Under some conditions, certain antiseptics are also effective disinfectants; they are effective
antimicrobial agents when applied to inanimate surfaces. Ethanol, for example, is categorized as
an antiseptic, but can also be a disinfectant. This depends on the concentration of ethanol used and
the exposure time, with disinfection generally requiring higher ethanol concentrations and
exposure times of several minutes. So, we can conclude that- all antiseptics are disinfectants but
all disinfectants are not antiseptics. (Brock 764)

Unusual Bacteria
1. Which bacteria is known as ‘sewage fungus’? Justify its name and role in sewage. (2020,
1.25 marks)
Ans: Sphaerotilus, a sheathed bacteria is known as ‘sewage fungus’. Its filaments are the main
component of a microbial complex that wastewater engineers call “sewage fungus,” a filamentous
slime found on the rocks in streams receiving sewage pollution. In activated sludge of sewage
treatment plants, Sphaerotilus is often responsible for a condition called bulking, where the tangled
masses of Sphaerotilus filaments so increase the bulk of the sludge that it remains suspended and
does not settle as it should. This has a negative effect on the oxidation of organic matter and the
recycling of inorganic nutrients and leads to treatment plant discharges with high nitrogen and
carbon loads. (Unusual Slide)
2. How are prosthecate and stalked bacteria suited to survive in their environment? (2020,
2 marks)
Ans: Prosthecate and stalked bacteria are appendaged organisms that attach to particulate matter,
plant material, or other microorganisms in aquatic habitats. Although a major function of these
appendages is attachment, they also significantly increase the surface-to-volume ratio of the cells
(prosthecae have large surface areas but almost no volume). The unusual morphology of
appendaged bacteria carries this theme to an extreme, and may be an evolutionary adaptation to
life in oligotrophic (nutrient-poor) waters where these organisms are most commonly found.
Prosthecae may also function to reduce cell sinking. Because these organisms are typically strict
aerobes, prosthecae may keep cells from sinking into anoxic zones in their aquatic environments
where they would be unable to respire. (Unusual Slide)
3. What would be a suitable microbiological medium and incubation condition for the
growth of myxobacteria? (2020, 0.5 marks)
Ans: Many myxobacteria can be grown in the laboratory on complex media containing peptone or
casein hydrolysate, which provides amino acids or small peptides as nutrients, and most are
obligate aerobes. (Unusual Slide)
4. Short note: (2019, 1.5*2=3 marks)
i. Myxobacteria
ii. Sheathed bacteria
Ans:
i. Myxobacteria: Typically, either long rods or filaments in morphology. Lack flagella but can
move when in contact with surfaces. The myxobacteria form multicellular structures called fruiting
bodies and show life cycles involving intercellular communication. The fruiting myxobacteria are
classified on morphological grounds using characteristics of the vegetative cells, the myxospores,
and fruiting body structure, and on phylogenetic grounds using 16S rRNA gene sequence analyses
to differentiate these different species of Deltaproteobacteria. (Unusual Slide)
ii. Sheathed bacteria: Sheathed bacteria are filamentous Beta-proteobacteria with a unique life
cycle in which flagellated swarmer cells form within a long tube or sheath. Under unfavorable
growth conditions, the swarmer cells move out and become dispersed to new environments,
leaving behind the empty sheath. Under favorable conditions, the cells grow vegetatively within
the sheath, leading to the formation of long, cell-packed sheaths. Sheathed bacteria are common
in freshwater habitats that are rich in organic matter, such as wastewaters and polluted
streams. Because they are typically found in flowing waters, they are also abundant in trickling
filters and activated sludge digesters in sewage treatment plants. In habitats in which reduced Iron
(Fe2+) or Manganese (Mn2+) is present, the sheaths may become coated with ferric hydroxide
Fe(OH)3 or various manganese oxides from the oxidation of these metals. (Unusual Slide)
5. Explain why the following bacteria are considered ‘unusual’ – (2018, 1.5*3=4.5 marks)
i. Spirochetes
ii. Sphaerotilus
iii. Hyphomicrobium
Ans:
i. Spirochetes: Spirochetes are slender, tightly coiled Gram-negative bacteria flexuous in shape
characterized by possession of endo-flagella used for motility. Spirochete motility is conferred by
flagella that emerge from each pole. However, unlike typical bacteria flagella, spirochete flagella
fold back from each pole onto the protoplasmic cylinder itself and remain in the periplasm of the
cell; because of this, they have been called endoflagella. In addition, both endo-flagella and the
protoplasmic cylinder are surrounded by a flexible membrane called the outer sheath. Spirochetes
have an unusual mode of motility. Each endoflagellum is anchored at one end and extends about
two-thirds of the length of the cell. Endoflagella rotate, as do typical bacterial flagella. However,
when both endoflagella rotate in the same direction, the protoplasmic cylinder rotates in the
opposite direction, placing torsion on the cell. This causes the spirochete cell to move by flexing
or lashing motions due to torque exerted at the ends of the protoplasmic cylinder by the rotating
endoflagella. (Unusual Slide)
ii. Sphaerotilus: Sphaerotilus filament is composed of a chain of rod-shaped
cells enclosed in a closely fitting sheath. Individual cells are 1–2 µm wide and 3–8 µm long and
stain Gram negatively. The cells within the sheath divide by binary fission, and the new cells
synthesize new sheath material at the tips of the filaments. Eventually, motile swarmer cells are
liberated from the sheaths that then migrate, attach to a solid surface, and begin to grow, with each
swarmer being the forerunner of a new filament. The sheath, which is devoid of peptidoglycan,
consists of protein and polysaccharide. Sphaerotilus cultures are nutritionally versatile and use
simple organic compounds as carbon and energy sources. Befitting its habitat in flowing waters,
Sphaerotilus is an obligate aerobe. (Unusual Slide)
iii. Hyphomicrobium: These organisms release buds from the ends of long, thin hyphae. The
hypha is a direct cellular extension and contains cell wall, cytoplasmic membrane, and ribosomes,
and can contain DNA. The mother cell, which is often attached by its base to a solid substrate,
forms a thin outgrowth that lengthens to become a hypha. At the end of the hypha, a bud forms.
This bud enlarges, forms a flagellum, breaks loose from the mother cell, and swims away. Later,
the daughter cell loses its flagellum and after a period of maturation forms a hypha and buds. More
buds can also form at the hyphal tip of the mother cell, leading to arrays of cells connected by
hyphae. (Unusual Slide)
6. Explain why Hyphomicrobium is able to adjust in different types of environments. (2018,
3 marks)
Ans: The main function of the myxospore is probably to allow the organism to survive desiccation
during dispersal or during intermittent drying of the habitat. Upon dissemination to a suitable
habitat or restoration of adequate growth conditions, the myxospore eventually germinates to form
a new vegetative cell. Many myxobacteria synthesize carotenoid pigments and thus their fruiting
bodies are brightly colored. Pigment formation is promoted by light, and at least one function of
these pigments is likely photoprotection, which would be beneficial in nature since the
myxobacteria are typically exposed to sunlight. In the genus Stigmatella, light greatly stimulates
fruiting body formation and catalyzes production of the compound 2,5,8-trimethyl-8-hydroxy-
nonane-4-one. This substance, a type of pheromone, promotes cell aggregation, the initial step in
fruiting body formation. (Unusual Slide)