Catalogue

TD 1 microbiology L2 staining in microbiology [Link] ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙1

TD [Link] culture media and sowing [Link] ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙5
TD 3 Microbiology [Link] ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙7
TD 4 measuring bacterial growth .pdf ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙11
TD 5 calculation of bacterial growth parameters .pdf ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙20
TD 6 TBAs L2 [Link] ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙22
TD 1 MICROBIOLOGY L2
Microscopic study and staining in microbiology

I- Microscopic study in microbiology :

Microscopic studies in microbiology enable us to study the morphology of microorganisms,
their shape, size, mode of association and sometimes their mobility.
It can be studied in two ways:
1- By preparing a fresh state: direct observation of our microorganism between slide and
coverslip from a preparation in liquid or solid medium.
To highlight: morphology, shape, size, mode of association and mobility
2- Preparing a smear :
A smear is a thin layer on a degreased slide used staining and subsequent microscopic
observation.
Preparing a smear :
• Spreading: spreading the bacterial preparation on a slide
• Drying: in the open air or with a Bunsen burner
• Fixation: run the slide through the Bunsen burner three times to fix the smear.
• Highlights: morphology, shape, size, mode of association, does not highlight
mobility

II- Staining in microbiology :

Microbiology stains can be :
1- Simple dyes: a single dye is used, e.g. methylene blue dye.
2- Differential dyes: several dyes are used (more than 1 dye)

Note: a smear is always used for staining, not the fresh state, with one exception (staining
with Indian ink to stain the capsule) (see below).
II.1- GRAM STAINING :

This is the most commonly used stain in microbiology, used to differentiate between
Gram-positive and Gram-negative bacteria. It is a differential stain because 3 dyes are
used: gentian violet, lugol and fuchsin. It is also known as alcohol-resistance staining.
Stages of colouring :
1- Take a smear on a slide, dry and fix with heat
2- Cover your slide with gentian violet for 1 min, this step is called the
colouring
 3- Rinse distilled water
4- Cover your slide with lugol for 30sec. This step is called etching because lugol helps the
gentian violet to bind better.
5- Rinse distilled water
6- Cover the slide with alcohol for 30 seconds. This step is called decolourisation and is
the most important stage of Gram staining.
7- Rinse distilled water
8- Cover the slide with fuchsin for 1 minute. This step is called counterstaining.
9- Rinse distilled water
10- Observation under the microscope.
Gram-positive bacteria appear in purple and Gram-negative bacteria appear in pink.

II.2- ziel-neeslson staining :

Used to stain mycobacteria, it is also known as Acid-alcohol resistance staining.
Colouring stage :
1- Taking a smear and fixing it
2- Colouring :
- Cover the slide with fuchsin and heat it with a Bunsen burner (20cm high so as not to
burn the smear). Heating should continue until white steam is emitted.
- Remove excess dye and allow the slide to cool, then rinse distilled water 3- Acid
resistance test:
- Cover the slide with nitric acid for 45 sec.
- Rinse distilled water.
- Cover the slide with 95% alcohol for 5 minutes
- Rinsing distilled water
- Cover the slide with methylene blue for 1 minute
- Rinsing distilled water
- Dry between sheets of absorbent paper
- Observation with a x100 microscope
Mycobacteria appear pink against a blue background.
II.3-Malachite green staining: this stain is used to identify bacterial spores.
Colouring stage
1- Taking a smear and fixing it
2- Colouring :
- Cover the slide with 5% malachite green, heat the slide using a ben bunsen
burner (20cm high so as not to burn the smear), heating should continue until
white steam is emitted, leave the slide to cool and repeat the same step three
times.
- Remove any excess dye and allow the slide to cool, then rinse distilled water.
- Cover the slide with 5% fuchsin for 1 minute.
- Rinsing distilled water
- Dry between sheets of absorbent paper.
- Observation with a x100 microscope
The bacterial spore appears green and the rest of the bacterial body pink.

NOTE: The spore can be identified using a negative methylene blue stain - the spore
appears colourless on a blue bacterial body.
II .4- Indian ink : This is the only stain where a fresh state is used instead of a smear, and
is used to highlight the bacterial capsule.
Colouring stage :
- Make a fresh state on a slide then add a drop of India ink
- Cover with a coverslip.
- Microscopic observation
The capsule appears as a clear transparent halo on a black background
TD 2: Culture media and inoculation techniques in microbiology
I) Growing media

A culture medium is a liquid or solid preparation for the growth, development and
identification of microorganisms. It must therefore meet the nutrient requirements (a
source of energy, carbon, nitrogen, essential ions, etc.) of the microorganism under study.

Culture media are classified according to :

1- There are three types of consistency:

☞ Liquid medium: does not contain agar agar, e.g. nutrient brothgrowth results in
cloudiness or superficial deposits and veils. Example: nutrient broth (BN)
☞ Solid medium: Solid media are obtained by adding a gelling agent to a liquid
medium. The most commonly used gelling agent agar-agar. As a general rule, to
transform a liquid medium into a solid medium, 15-18 g of agar-agar must be
added to 1 L of liquid medium, e.g. nutrient agar, which is a nutrient broth+ 15 g of
agar-agar.
☞ Semi-solid medium: containing (7.5-9 g.L-1) % agar agar, e.g. Muller Hilton MH
medium

2- Depending on their use: In general, there are 4 main types of media:

• Isolation media: This is a basic medium, allowing growth of all bacteria, e.g.
nutrient agar.

• Selective medium :
Designed to encourage the growth of certain bacteria compared with others (encourages
the growth of certain bacteria and prevents the growth of other micro-organisms. To
achieve this, elements are added to inhibit the growth of undesirable micro-organisms,
such as NACL in Chapman medium: Chapman is a medium containing 75 g of NaCl,
which encourages the growth salt-loving bacteria (halophilic bacteria) and inhibits
the growth of bacteria that cannot tolerate salt.

• ENRICHED MEDIUM :
Is a basic medium enriched with growth factor (a substance that promotes the growth of
certain bacteria, e.g. blood agar), is a basic medium that is nutrient agar enriched with
blood (blood is a source of iron, so blood agar is an iron-enriched basic medium).

• Differential or identification medium

The so-called differential culture medium or indicator identification medium is used to
distinguish between two types of microorganism growing in the same medium. This type
of medium highlights
to highlight certain biochemical characteristics of the microorganisms (mainly the ability
to degrade a substrate) in the presence of indicator(s) of the chemical reaction: coloured
indicators of pH or oxidation-reduction, e.g. Chapman medium is a differential medium
that also makes it possible to differentiate between different halophilic bacteria using the
colour change linked to the degradation of mannitol.

Conservation environments: these are poor environments in which bacteria survive in a

state of slowed life.

II) Seeding techniques :

Liquid-liquid inoculation: passage of the bacterial inoculum from a liquid medium to a

new liquid medium.
Liquid-solid streak plating: The inoculum is deposited on an agar medium, then the
platinum line or the loop of the pasteur pipette is carefully wiped over the surface of the
agar. This method is used when the agar is poured into petri dishes.

Solid-liquid plating Dissociation in a liquid medium of a bacterial colony collected with

the platinum loop.

by flooding: The surface of the agar medium in a petri dish is flooded with the inoculum
and spread evenly using a sterile spreader or rake. The excess inoculum is then sucked off
and left to dry in an oven for a few minutes at 37°C (e.g. antibiogram test).

Inoculation into the mass: In this method, the inoculum is mixed with the agar medium
before it solidifies. This method is used for bacterial enumeration.

Spot plating: the concentrated inoculum is deposited on a small area medium (a few
millimetres in diameter). This technique allows several culture trials to be carried out on
the same dish (e.g. testing several strains).

Inoculation by central pricking: this is generally done on tubes containing an agar

medium by introducing the pasteur pipette loaded with inoculum vertically at the bottom
of the tube. This technique is used to demonstrate the mobility of the inoculum.
 TD 3: sterilisation methods in microbiology

I. Key concepts:

• Sterilisation: is the action of killing all forms of microbial life contained in a

preparation or present on the surface an object. The material treated is said to be
sterile when no micro-organism can be revived. Objects often need to be packaged
before sterilisation and preparations need to be tightly sealed to prevent subsequent
contamination. Sterilisation can also be defined as the operation that consists of
eliminating micro-organisms from an object, in a lasting and definitive manner.

• Disinfection: is a measure designed to destroy pathogenic microbes. It is

ineffective on endospores. It uses a chemical called a disinfectant on inert products.
This is a temporary operation. It destroys the microbes present, but the treatment
must be repeated in the event of subsequent contamination. If it is applied to living
tissue, it is called antisepsis and the product used is an antiseptic. The same
products are not used on living tissue and inert objects.
The antiseptic must be non-toxic and non-irritating humans or animals.

• Decontamination: like disinfection, decontamination is an action with non-

permanent results. It inhibits micro-organisms, without necessarily killing them.
It is applied to living tissue.

• Asepsis is the set of rules to be observed by medical teams to prevent the

introduction of exogenous microbes.
II. Sterilisation methods :

II.1. Sterilization by heat: Thermal sterilization uses moist heat or dry heat to
destroy micro-organisms (bacteria and spores). There are two methods (dry heat
sterilization and moist heat sterilization).

II.1.1 -Dry heat sterilisation: Heat is used to sterilise

Flaming: Passing the surface of non-flammable material through a flame (Bunsen burner)
ensures good sterilisation. Platinum wires and Pasteur pipettes are sterilised in this
way.

Sterile zone of a Bunsen burner

Le Four pasteur :
This is a dry hot-air oven. It operates at 180°C for 90 minutes. This device is only used
to sterilise glassware that has been cleaned, dried and packaged.
The sterilised equipment is left to cool completely, then stored in a dust-free place.

II.1.2. Sterilisation by moist heat; these methods use heat+ and pressure to sterilise.

1. The autoclave is an essential piece of equipment in a microbiology laboratory.

Autoclaving is a method of sterilisation that involves heating under steam
pressure, which is equal to 1 bar, at a temperature of 121°C for 20 minutes. This
process kills all the vegetative cells and endospores.

2.
2. Tyndallisation: Used to sterilise fragile materials that can be altered by heat.
Tyndallisation is a series of 3 brief heatings at temperatures of 60-70°C at regular
intervals, separated by rest periods at the ordinary temperature, in order to give the
resistant forms the opportunity to germinate and kill them when the next heating takes
place (endospores that have reactivated into vegetative cells).
Example: milk pathogens are destroyed by a cycle of 63°C for 30 minutes followed by
73°C for 15 minutes.

3. Pasteurisation
Pasteurisation is a heat treatment that applies a temperature of 63.5 for 30 minutes or
75°C for 15 minutes (the higher the heating temperature, the shorter the exposure time).
Pasteurisation temperatures range from 63.5 to 85°C followed sudden cooling to 4°C
(sudden cooling kills the spores):
-High temperature pasteurisation: The milk is heated to 90°C for 30 seconds and then
cooled to 10°C.
Low-temperature pasteurisation: Heating to 60-70°C for longer periods.
-Ultra High Temperature (UHT) pasteurisation: The most recent method, it is applied
to milk and fruit juice. A temperature of 140°C is reached for a few seconds, followed by
sudden cooling to eliminate the spores.

II.2. Mechanical sterilisation: example of filtration:

Filtration is the best way to sterilise solutions containing heat-labile substances, such as
proteins, vitamins, serum, TBAs, etc. Sterilising filtration is known as cold sterilisation.
This technique involves passing a liquid through a filter whose pores have a diameter of
0.1 μm. The micro-organisms are too large and are therefore retained by the filter. This
technique is the best way of sterilising solutions containing thermolabile (temperature-
sensitive) substances, such as proteins, vitamins, serum, TBAs, etc. Sterilising filtration is
known as cold sterilisation. There are different filtration systems depending on the
volumes treated.
II.3. Other sterilisation processes

• UV radiation :
UV light is used to sterilise laboratory equipment, such as micropipettes ....ect, acting on
DNA by chemically modifying or cutting it.
 TD 4: MEASURING BACTERIAL GROWTH
Introduction :

Microbial growth is defined as the increase in cell constituents, and can also be expressed
as an increase in cell size, cell number or both.

It can be defined as the orderly growth of all the components an organism, such as
bacteria (a single-celled organism), where growth results in an increase in the number of
individuals.
Other division mechanisms exist in particular bacteria, such as budding (observed in
cyanobacteria) and fragmentation (in filamentous bacteria).
The point of division is called the septum, which will form the partition separating the two
daughter cells. Each cell will receive a copy of the chromosome and half of the cellular
components. In bacteria, division occurs by scissiparity, giving us two daughter bacteria.
The study of bacterial growth involves determining the growth parameters for a given
bacterial strain, which are :
1- Generation time: this is the time required for the number of bacteria to double.
E. coli : 20min
2- Growth rate: this is the number of divisions per unit of time E coli: 3 times / hour
Some examples of bacteria:
Time to
Species Growth rate 1/G (h-1)
generation G (min)
Vibrio paraheamolyticus 10 6
Escherichia coli 20 3
Lactobacillus acidophilus 100 0,6
Mycobacterium tuberculosis 1000 0,06

1. Measuring growth :

1.1 Indirect viable cell count (Enumeration of bacteria after culture)

1.1.1. Enumeration in solid media :
Counting viable bacteria is a commonly used method. A fixed volume (0.1 to 1ml) of
the stock culture or decimal dilutions (prepared from the stock solution, 1ml of milk,
juice or a few mg of ground meat, in 9ml of sterile water (first dilution 1/10).
A serial dilution of 1/100, 1/1000, 1/10000, 1/100000, 1/1000000 is then performed.
Then 1 ml is spread on the surface of an agar medium or incorporated into the
medium before it solidifies (in depth).
After incubation at a suitable temperature (37° for 24 hours), the number of colonies
that appear corresponds to the number colony-forming units (CFU) present in the
volume of suspension analysed. The suspension must be homogenous by mechanical
agitation. This is referred to as colony-forming units (CFU). Each colony comes from
several bacteria.

∑ C: number of colonies counted on the set of boxes retained (positive boxes) n:

number of positive boxes (number of boxes retained in the number varies between 30
and 300)
n1: number of cans counted at the lowest dilution
n3: number of cans counted at the highest dilution
nn: number of boxes retained (positive from the nth dilution)
vn: volume of inoculum from the nth dilution
dn: number of the nth dilution Fd:
initial dilution factor (d1)
The number of colonies retained after reading the plates should be between 30 and
300. A plate containing more than 300 colonies cannot be taken. The risk of error is
too great. Boxes containing fewer than 30 colonies are not included in the calculation,
as the colonies are too rare and could be .
30 * N 300

+1 ml Milk

10' 10'

Sample 9 ml 9 ml 9ml

N
Zc
((ni x v,)dl -1- ( 2 X Vz)d2+ ( 3 X V3)d3)
Dilution 10" 10" 10'"
Box 1
Box 2
Box 3

*C
N --
((ni x v,)dl+ (n2 x v )d2+ (n3x v,)d3)

300+ 270+ 250+ 87+ 90+ 60

N --
((3 x1)10°')+ (3 x1) 10°'))
300 270 -F 250 -F 87 -F 90 60
N --
((3 x 1)+ (3 x 10°'))10"
1057
N- = 348 . 10' UFC/ml
(3+ 0.3)10*'
Surface -' 0.1 ml -' rake
(mo yene des 3 boites)
N --
ri P d'Înocu/un . Fd
300+ 270+ 250
N -- = 273 . 10' CFU/m1
3x0.1 x 10*'
1.2 Liquid-based enumeration (Mac Grady's MPN method) :
This is a statistical method used to count microorganisms in a liquid medium. The
water sample or its dilutions are incorporated into a liquid culture medium designed
to allow the growth of a microorganism or group of microorganisms. Growth results
in the appearance of a turbidity in the medium, which may be accompanied by the
appearance of gas and, possibly, a visible change (turning of a coloured pH
indicator). It is generally used to detect and count total coliforms, faecal coliforms
and faecal Streptococci in water.
Make a dilution of a given volume of the sample to be analysed, inoculate 1ml of each
dilution in several 2.3.4 tubes (e.g. take 3 tubes for each dilution). For each
series from the same dilution, count the number of positive tubes and record the
results in a table to determine the characteristic number.
N: number of micro-organisms per ml of analysed product or parent suspension.
NPP: the number read from the Mac Grady table.
Fd: dilution factor corresponding to the hundreds digit of the characteristic number.
V: volume of inoculum (usually 1ml)
Calculating number of bacteria in example 1 in the table :
Choice of dilution: 10-4
hence Fd=dilution factor= 104
Determination of the MPN: grouping chosen = 321 and, in the Mac Grady table, the
MPN corresponding 321 is 15. This means that there are statistically fifteen bacteria
in the 10-4 dilution inoculum.
.
Inoculated volume= 1 ml

N = 15/1 ×10 4 = 15 .104 Germs / ml

1.3 Turbidimetric method (measurement optical density Do):

This technique is simpler and is widely used to count microbial cells. by

measuring the turbidity of the culture, hence the name turbidimetry. The more a
bacterium grows in a liquid, the more turbid the liquid becomes. A haze is formed.
This technique is based on measuring the absorbance of incident light in relation to
transmitted light. Do 600nm for bacteria. A tube containing a bacterial suspension is
placed in a spectrophotometer, the emitted by the source passes through the tube,
part of this light is absorbed and the other part transmitted is captured by the
detector.
Under precise technical conditions, absorbance is proportional to cell concentration.
The disadvantage of this technique is that it cannot be used in highly coloured media,
and it is unable to differentiate between dead and healthy cells.

Figure 01. Determination of turbidimetry by spectrophotometer

2. Growth parameters :
These parameters are also known as growth constants.
Cell division follows an exponential progression at regular times. 01 cell gives 02 cells,
which give 04 cells, then 16, then 32 and so on.
The time required to double the number of cells is called the generation time (G).
Generation time is specific to each species and depends on environmental conditions.
(G) is 20 minutes for Escherichia coli, 1000 minutes for
Mycobacteriumtuberculosis.

3. Growth curve

3.1. Growth curve in non-renewed medium, discontinuous culture

In a discontinuous culture, where growth is not synchronous and nutrients are depleted over
time, growth follows an 04-phase curve. The latent phase, the exponential growth phase,
the stationary phase and the decline phase.
 Figure 02; The bacterial growth curve

3.1. The latency phase

The growth rate is zero. Bacteria do not divide, but adapt to the conditions of their
environment. They synthesise the necessary enzymes specific to the substrates (nutrients)
present. They increase in size and prepare to divide.
The duration of this phase varies according to the nature of the inoculum: if the culture is
fresh (young culture 16h) and comes from the same medium (short phase), if the culture is
old or comes from a different medium it will be longer.
If you inoculate the same medium with bacteria taken during the exponential phase, there
will be no latency period. This is an undesirable stage in the industry and tanks are used to
avoid it.

3.2. The exponential growth phase

Bacterial cells divide continuously, as long as nutrients are available, toxic substances are
absent and the pH is optimal. The rate of growth is maximal and constant. The
physiological state is also maximal. The bacteria will divide, using the nutrients present in
the environment until the substrates are exhausted, while at the same time producing toxic
substances that will change the physico-chemical characteristics of the environment. This
is the phase when the growth rate and generation time are calculated.
Industry avoids the end of the expo phase and the beginning of the stationary phase, at
which level waste accumulates, especially if you want to extract a metabolite such as
penicillin, the waste will interact with it.
3.3. The stationary phase
When culture is carried out in a flask or tube, at some point the nutrients run out, toxic
products accumulate and the pH changes. The number of cells no longer varies. There is as
much cell division as cell death. The growth rate (u) is constant. There even talk of a
cryptic growth, characterised by cell lysis of some cells but not all, or cells will take up the
substrate released during lysis and use it to divide.
There is cell lysis and production of new replacement cells, with constant growth.
3.4. The decline phase
Bacteria no longer divide. They die by cell lysis. The growth rate (u) is negative.

3.2. Phenomenon of diauxia (two growths in Greek)

The phenomenon of diauxia is a growth that takes the form of a biphasic curve. This
growth is observed when a synthetic medium containing two carbon sources is used. In a
medium containing glucose and lactose, the bacteria will use the glucose first, thanks to its
constituent enzymes. Lactose degradation is dependent on inducible enzymes whose
induction is repressed in the presence of glucose. When the glucose is consumed, the
bacteria will use the lactose and initiate a new phase of exponential growth after an
adaptive latency period.

Figure 03: Diauxie phenomenon (Phase I (Glucose) -Phase II (Lactose))

Growth continues

The four growth phases seen so far (latency, exponential, stationary and decline) are
obtained in a closed medium. To achieve a goal of industrial interest (e.g. fermentation), it
is more practical to maintain the bacteria in an exponential phase. This can be achieved by
renewing the culture medium and eliminating the products of metabolism using
Turbidostats (Figure 07) and Chemostats.
Figure 04: Turbidostat diagram
 TASK 5: CALCULATION OF BACTERIAL GROWTH PARAMETERS

Exercise 1:
A bacterial growth experiment is carried out with a strain of [Link] that requires guanine
as a growth factor. The experiment is performed at 37°C using a 24-hour culture in a
medium containing guanine.
The variations in the number of bacteria per ml of culture medium as a function time are
studied, where N is the number.

Time 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
(h)
Log10N 2.0 2.0 2.0 2.2 2.8 3.5 4.4 5.3 6.1 6.8 7.0 7.0 7.0 7.0 7.0 6.9 6.5 5.7

1. Plot the representative curve of the log10 function N = f (t)

2. Characterise each phase E. coli growth in the medium used.
3. Calculate the growth rate during the exponential phase. Deduce the value of the
generation time during this same phase. Take log 10 (2) = 0.3

Correction:
1. The curve will be drawn by the students beforehand
2. Characterisation of growth phases:
Time (0 - 2h) the latency phase Time (3-
10 h) the exponential phase Time (10-
14h) the stationary phase
Time (15- 17) the decline phase

3. Calculate the growth rate during the exponential phase:

µ = Log Nn- log N0 / (t-t0) Log2 µ =6.8-5.3 / (9-7) ×0. µ =3divisons

/h Deduction of generation time: G= 1/ µ = 1/ 3= 20min

Exercise 2:

Bacillus cereus is a bacterium that divides every 30 minutes. A culture is inoculated

with 100 bacteria.
How many bacteria will be present after 3 hours incubation.

Correction:
Bacillus divides every 30 minutes, in 3 hours there will be 6
divisions, n =6 Nn =2n × N0
N3h= 2 ×100
6

N3h= 64×100 = 6400 bacteria.

Where
µ = log Nn- log N0 / (t-t0) log2
Where
Log Nt= log N0 + n log 2
 TASK 5: CALCULATION OF BACTERIAL GROWTH PARAMETERS

Exercise 3:
A liquid medium was seeded with 50 bacteria after n generations and 512 103 cells
were numbered.
Determine the number of generations n.

Correction:
N0= 2 50
0

N1= 50×2
N2= (50×2) ×2
N3 =(50×2×2)×2
Nn =2n× N0

Nn =2n× N0
Nn /N0=2n
Ln Nn / Ln N0 = Ln 2n
Ln Nn - Ln N0 = n Ln 2
N= (Ln Nn -Ln N0) / Ln2
n= (Ln 51.2 103- Ln50) /Ln2
n=10

Exercise 4:
If an exponential phase culture contains 100,000 cells / ml at one point and after
4 hours the population is 100,000,000 cells / ml, what would be the μ and g
values?

u= logNn-logN0 / Δt log 2

N0= 10000 cells /ml

N= 100,000,000 cells/ml.
Δt= 4h

u= 2.303 h-1

Calculation of G

G= 1/u = 1/ 2.303= 0.3 h

Definition a TBA :

Substance capable of specifically inhibiting the growth of micro-organisms or destroying them. The latter may be a
chemical substance produced by micro-organisms or obtained by semi-synthesis or chemical synthesis.

History :

1927: Fleming discovers penicillium on a Petri dish (inhibition of staphylococcus aureus)

• 1940: Chain obtains a stable solution penicillin (1st antibiotic)

• 1942: industrial production of penicillin (used during Second World War)

Preliminary definition :

Chemotherapy :

This is the use of chemical agents in therapy. To be used in treatment, the substance must be harmful.
for the parasitic microorganism but harmless to the host cell - this is the case with TBAs and sulphonamides.

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Antibiotics are antimicrobial agents. They are chemicals, synthetic or natural, used within the host's tissues to destroy or
inhibit pathogenic micro-organisms. These chemotherapeutic agents are non-toxic.
Depending on their antimicrobial action, there are two categories antimicrobial agents:
- Static antibiotics: Agents that inhibit growth.
Ex: bacteriostatic and fungistatic.
- Germicidal antibiotics which completely destroy pathogenic germs, but not necessarily the endospores. E.g.
bactericides, fungicides, algicides and virucides.
Antibiotics can also be classified according to their spectrum of activity, in which case two groups antibiotics are
distinguished: broad-spectrum antibiotics and narrow-spectrum antibiotics.
Chemical classification is the most commonly used, based on the chemical composition of each antibiotic (Table 6).
In addition, mode and site of action of antibiotics enable them to be classified.

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 1. Mode of action
The targets usually targeted by the action of antibiotics are those which constitute an essential part of the body's
immune system.
and constant for the survival of the bacterial cell.
The bacterial wall, membrane, DNA and ribosomes are all sites antibiotic action (Figure 22).

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Antibiotic susceptibility test: laboratory test used assess the sensitivity of a bacterium found.
after culture a patient sample against various ATB in vitro (on petri dishes)

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• Bacteriostatic: stops bacteria from multiplying

Minimum Inhibition Concentration MIC: The lowest concentration of antibiotic that inhibits any
visible culture a bacterial strain after 18 hours of culture at 37°C. This value characterises the
bacteriostatic effect of an antibiotic.
• Bactericide: kills bacteria

Minimum Bactericidal Concentration (MBC): The lowest concentration antibiotic that

kills 99.99% of a bacterial population in 18-24 hours.

Minimum Bactericidal Concentration (MBC): smallest concentration of antibiotic leaving

0.01% or less survivors of the initial inoculum after 18 hours of culture at 37°C. This value
characterises the bactericidal effect of an antibiotic. The same TBA can be bacteriostatic at a low dose
and bactericidal at a higher dose.

• Activity spectrum: capacity to have an activity on a defined micro-organism

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Bacteriostatic antibiotics - Bactericidal antibiotics Antibiotics are classified according to their activity:
Bacteriostatic antibiotics BMC far from MIC: BMC> 32 x MIC
macrolides, tetracyclines, rifamycins, sulphonamides
Bactericidal antibiotics: BMC close to MIC: BMC < 32 x MIC
aminoglycosides - β-lactams - quinolones - glycopeptides

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c- According to the results obtained, is the antibiotic amoxicillin bactericidal or bacteriostatic for E. coli? Justify. (1 pts: see
the breakdown in the answer) The antibiotic amoxicillin is bactericidal for E. coli because survival after 24 hours is 0.00062
% (i.e.
<0.01). Remember: an antibiotic said to bactericidal when it kills more than 99.99% bacteria, i.e. 0.01% survival rate.
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2.49 109 100%

1.56 104 X

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