MEDICAL MICROBIOLOGY I

Lecture 1
Laboratory Safety and Bacterial
Growth

Laboratory Safety
Identify and evaluate hazards
Type of hazard:
1. Biohazards (infectious agents or items
contaminated with them)
2. Irritants (cause reversible inflammation)
3. Corrosive chemicals
4. Sensitizers (cause allergic reactions)
5. Carcinogens (induce tumours)

Laboratory Safety Guidelines

Wash hands with disinfectant soap when you
arrive at the lab and again before you leave.
No eating, drinking, chewing gum, or smoking in
the lab.
Do not put anything in your mouth such as pencils,
pens, labels, or fingers.
Avoid loose fitting items of clothing.
Application of cosmetics other than hand lotion
likewise has no place within laboratory setting.
Keep your workspace free of all unnecessary
materials.

Laboratory Safety Guidelines

Disinfect work areas before and after use with
70% ethanol or other disinfectant.
Label everything clearly.
Replace caps on reagents, solution bottles, and
bacterial cultures.
Do not open Petri dishes in the lab unless
absolutely necessary.
Inoculating loops and needles should be flame
sterilised in a Bunsen burner before you lay them
down.

Laboratory Safety Guidelines

Turn off Bunsen burners when not in use.
Solutions must never be pipetted by mouth.
Treat all microorganisms as potential
pathogens. Use appropriate care and do not
take cultures out of the laboratory.
Do not pour anything down the sink.
If hazardous powders have been handled, wash
around your nose and mouth so that adherent
particles are not ingested or inhaled.

Labeling
The solutions created in laboratory must
be fully labeled.
Minimum information:
Chemical name and, if a mixture, names of all
ingredients
Manufacturers name and address if purchased
commercially, or person making the reagent
Date of purchase or made
Expiration date, if known
Hazard warnings and safety precautions

Personal Protective Equipment

Clothing suitable for laboratory work should be
considered before protective equipment.
1. Secure, close-toed footwear
2. Lab coat

prevent biohazardous materials from reaching

skin, and more importantly, any cuts, dermatitis,
etc., that may be present
also protect street clothing from needing
decontamination, as well as preventing
contamination of laboratory cultures from the
normal flora present on the skin

Personal Protective Equipment

Personal Protective Equipment

1. Aprons - disposable; heavy rubber aprons may be
needed when handling concentrated acids
2. Gowns - prevent biohazardous materials from
reaching skin and prevent contamination of
laboratory cultures
3. Goggles or safety glasses to protect the eyes by
preventing splashes into the eyes, nose and mouth,
and onto the skin
4. Full-face shields should be worn to protect the eyes,
nose and mouth, and the facial skin

Personal Protective Equipment

5.

Gloves
prevent exposure to microorganisms
glove material is rarely completely
impermeable; it delays penetration of harmful
material for a time sufficient to provide
adequate protection
latex gloves are suitable
only for protection from
biohazards

Personal Protective Equipment

For the best protection, the
cuffs of the gloves should
overlap the lower sleeves of
the laboratory coat
Exam gloves must not be
reused. They are designed for
disposal after one use or if
exposed to a chemical (they
offer limited chemical
protection).

Personal Protective Equipment

6. Surgical masks-basic protection
7. Respirators
e.g. HEPA (high efficiency particulate air) maskbetter protection
prevent the inhalation of aerosolized
microorganisms (inhalation exposure) when safety
equipment designed to contain infectious aerosols,
such as a biosafety cabinet, is not available

Specimen Handling
There are 3 potential routes of exposure:
1. Inhalation of aerosols
2. Contact with skin
3. Contact with mucous membranes (eyes, nose and
mouth)

Fresh specimens of human origin MUST always

be considered potentially INFECTIOUS.
Fixed specimens have much-reduced risk
because almost all infectious agents are
deactivated by histological fixation.

Control of Hazardous Material

Proper control of hazardous material involves:
1. Collection
2. Transportation
3. Disposal / Decontamination

Collection
Biohazardous material
Place discarded material in biohazard waste
container (lined with yellow biohazard bag).
Place used syringe / needle / glass slide / lancet
/capillary tube / broken glass fragments into
biohazard sharp container.
Discarded used microbiology plates must be
disposed in a biohazard container filled with
sodium hypochlorite.

Transportation
Laboratory personnel are responsible for the
packaging of biohazardous / pathological waste.
If waste packaged in biohazard bags, have to be
sealed / tied
If waste are in container (biohazard / sharp), have
to be closed and secured shut
All biohazard bags and container have to be labeled
with the generators (lab) name, date and time.

They are also responsible for transporting the

waste to the designated collection points.

Disposal / Decontamination
Depending on the material, there are several
means by which items can be treated.
The most common methods of treatment and
disposal are:
Disinfection using chemicals
Sterilization using steam (autoclave)
Incineration (burning at high temperature)

Disinfection
Categorised as:
1. High level
2. Intermediate level
3. Low level

Effectiveness is influenced by
the nature of the item to be disinfected
number and resilience of the contaminating organism
amount of organic material present
type and concentration of disinfectant
duration and temperature of exposure

Disinfection
1. High-level disinfectants
e.g. moist heat, and use of liquids such as
glutaraldelyde, hydrogen peroxide, peracetic acid,
chlorine dioxide, and other chlorine compounds
Involve invasive procedures (e.g. certain types of
endoscopes, surgical instruments with plastic or other
components that cannot be autoclaved)
Cleaning the surface to remove organic material
Cleaning surface that are exposed to resilient
organisms and bacterial spores

Disinfection
2. Intermediate-level disinfectants
e.g. alcohols, iodophor compounds, phenolic
compounds
Semi-critical instruments and devices such as flexible
fiber-optic endoscopes, laryngoscopes, vaginal
specula, and anesthesia breathing circuits.
Clean surface or instruments in which contamination
with bacterial spores and other highly resilient
organisms is unlikely.

Disinfection
3. Low-level disinfection
e.g. quaternary ammonium compound
Used to treat non-critical instruments and devices
such as blood pressure cuffs, electrocardiogram
electrodes, and stethoscopes
Although these items come into contact with
patients, they do not penetrate through mucosal
surfaces or into sterile tissues.

Disinfection
The level of disinfectants used for environmental
surfaces is determined by the relative risk these
surface pose as reservoir for pathogenic
organisms.
High-level disinfectant should be used to clean
the surface of instruments contaminated with
blood
Not to clean surfaces that are dirty such as
floors, sinks, and countertops.
Exception to this rule is if a particular surface has
been implicated in nosocomial infection.

Disinfection
Chemical disinfection can also be achieved
using gas.
The most common example is the use of
ethylene oxide.
Gas disinfection is advantageous when the
sample is such that scrubbing of inner surfaces
cannot be done, such as in tubing.

Sterilization
Steam under pressure in an autoclave is a very
effective form of sterilization.
High temperature (121 - 135C) for 15 min
causes denaturation of microbial proteins.
Rapid rate but is influenced by:
Temperature and duration of autoclaving
Size of the autoclave
Flow rate of the steam
Density and size of the load
Placement of load in the chamber

Incineration
Practiced routinely in microbiology laboratory
to sterilise the inoculating loops.
Exposing the inoculating loop to a gas flame
will burn up and vaporise any living microbes
that are on the loop, ensuring that infectious
organisms are not transferred.
Incineration is carried out in specially
designed furnaces that achieve high
temperatures and are constructed to be
airtight.

Incineration
Proper incineration should:
occur very quickly
should not leave any residual material.
be smoke-free, otherwise microbes that are still
living could be wafted away in the rising smoke
and hot air to cause infection elsewhere.
not too much sample as can result in an
incomplete burn.

Bacterial Growth
Bacterial growth is the division of one
bacterium into two daughter cells in a process
called binary fission.
Providing no mutational event occurs the
resulting daughter cells are genetically
identical to the original cell.
If the number surviving exceeds unity on
average, the bacterial population undergoes
exponential growth.

Bacterial Growth
1. Lag phase
When microorganism are introduced into fresh culture
medium, usually no immediate increase in cell
number.
Cells synthesising new components
The cells may be old, depleted of ATP, essential
cofactors, and ribosomes - all must be synthesised
before growth can begin
New enzymes would be needed to use different
nutrients.
Cells might be injured and need time to recover.
Inoculation of culture into a chemically different
medium also results in a longer lag phase.

Bacterial Growth
Exponential or log phase
Microorganisms are growing and dividing at their
maximal rate and are influenced by:
Genetic potential
Nature of the medium
Conditions under which they are growing

Rate of growth is consistent, so there is balanced

growth.
The population is most uniform in their chemical and
physiological properties.
Usually used in biochemical and physiological studies.

Bacterial Growth
Stationary phase
In a closed system, eventually population growth
ceases and the growth curve becomes horizontal.
Attained by bacteria at a population level of around
10 cells / mL.
Final population size depends on:
nutrient availability
oxygen (other gases) availability
type of microorganism being cultured

Bacterial Growth
Stationary phase
Total number of viable microorganisms remain
constant due to:
balance between cell division and cell death, or
the population may simply cease to divide but remain
metabolically active.

Limiting factors
Nutrient
Oxygen / Other gases

Bacterial Growth
Senescence and Death
Assumed that detrimental environmental changes like
nutrient deprivation and the buildup of toxic wastes
caused irreparable harm resulting in loss of viability.
Even when bacterial cells were transferred to fresh
medium, no cellular growth was observed.
Loss of viability was often not accompanied by a loss
in total cell number, it was assumed that cell died but
did not lyse.
Viable but non-culturable (VBNC)
Programmed cell death or apoptosis

Growth of Microorganism
Several factors influence the growth of
microorganism:
1.
2.
3.
4.
5.

Nutrition
Oxygen
pH
Temperature
Moisture

Nutrition
Enters the cell after passing across the cells
membrane.
Used by the cell for building material, cellular
synthesis or for obtaining energy.
Nutritional requirements of bacteria varies
among species.
Some microorganism can obtain all their
nutritional requirements from inorganic matter
while others need many complex organic
compounds.
Requirements: carbohydrates (sugar, starches and
cellulose), a source of nitrogen, vitamins, water, and a
source of energy

In addition to a proper physical

environment, microorganisms also
depend on an available source of
chemical nutrients and are usually
grouped according to their energy and
carbon sources

Energy Source
1. Phototrophs
Use radiant energy (light) as their primary energy
source.

2. Chemotrophs
Use the oxidation and reduction of chemical
compounds as their primary energy source.

Carbon Source
Carbon is the structural backbone of the organic
compounds that make up a living cell.
Based on their carbon sources, bacteria can be
classified as:
1. Autotrophs
Require only carbon dioxide as a carbon source. An
autotroph can synthesise organic molecules from
inorganic nutrients.

2. Heterotrophs
Require organic forms of carbon. A heterotroph cannot
synthesise organic molecules from inorganic nutrients.

Nutritional Pattern
Combining their nutritional patterns, all
organisms in nature can be placed into one of
four groups:
1.
2.
3.
4.

Photoautotrophs
Photoheterotrophs
Chemoautotrophs
Chemoheterotrophs

Photoautotroph
Photoautotrophs use light as an energy source
and carbon dioxide as their main carbon
source.
They include photosynthetic bacteria (green
sulfur bacteria, purple sulfur bacteria and
cyanobacteria), algae, and green plants.
Photoautotrophs transform carbon dioxide
and water into carbohydrates and oxygen
through photosynthesis.

Photoheterotroph
Photoheterotroph use light as an energy
source but cannot convert carbon dioxide into
energy.
Instead they use organic compounds as a
carbon source. They include the green nonsulfur bacteria and the purple non-sulfur
bacteria.

Chemolithoautotroph
Chemolithoautotrophs use carbon dioxide as
their main carbon source and inorganic
compounds as an energy source.
Among the inorganic compounds are:
hydrogen sulfide
Sulfur
Ammonia
Nitrites
hydrogen gas
iron

Chemoorganoheterotroph
Chemoorganoheterotrophs use organic
compounds as both an energy source and a
carbon source.
Saprophytes live on dead organic matter while
parasites get their nutrients from a living host.
Most bacteria, and all protozoas, fungi, and
animals are chemoorganoheterotrophs.

Nutrition
Minerals
1. Sulfur
Needed to synthesise sulfur-containing amino acids
and certain vitamins.
Depending on the organism, sulfates, hydrogen
sulfide, or sulfur-containing amino acids may be used
as a sulfur source.

2. Phosphorus
Needed to synthesise phospholipids, DNA, RNA, and
ATP.
Phosphate ions are the primary source of
phosphorus.

Nutrition
3. Potassium, magnesium, calcium
Required for certain enzymes to function as well as
additional functions.

4. Iron
Part of certain enzymes

5. Trace elements
Required in very minute amounts
Usually function as cofactors (electron donors or
electron acceptors) in enzyme reactions
e.g. sodium, zinc, copper, molybdenum, manganese,
and cobalt ions.

Oxygen
Microorganisms show a great variation in their
requirements for gaseous oxygen.
Most can be placed in one of these groups:
1. Obligate aerobes
Organisms that only grow in the presence of oxygen.
Obtain their energy through aerobic respiration

2. Microaerophiles
Organisms that require a low concentration of
oxygen (2-10%) for growth, but higher concentration
are inhibitory.
Obtain their energy through aerobic respiration.

Oxygen
3. Obligate anaerobes
Organisms that grow only in the absence of oxygen
and are often inhibited or killed by its presence.
Obtain their energy through anaerobic respiration
or fermentation

4. Aerotolerant anaerobes
Like obligate anaerobes, they cannot use oxygen to
transform energy but can grow in its presence.
Obtain energy only by fermentation and are
known as obligate fermenters

Oxygen
5. Facultative anaerobes
Organisms that grow with or without oxygen, but
generally better with oxygen.
Obtain their energy through aerobic respiration if
oxygen is present, but use fermentation or
anaerobic respiration if it is absent.
Most bacteria are facultative anaerobes.

pH
Microorganisms can be placed in one of the
following groups based on their optimum pH
requirements:
1. Neutrophiles

Grow best at a pH range of 5 to 8

2. Acidophiles

Grow best at a pH below 5.5

3. Allaliphiles

Grow best at a pH above 8.5

Temperature
Bacteria have a minimum, optimum, and
maximum temperature for growth and can be
divided into 3 groups based on their optimum
growth temperature.
1. Psychrophiles
Cold-loving bacteria.
Their optimum growth temperature is between 5 and 15C.
They are usually found in the Artic and Antarctic
regions and in streams fed by glaciers.

Temperature
2. Mesophiles

Bacteria that grow best at moderate temperatures.

Their optimum growth temperature is between 25 45C.
Most bacteria are mesophilic and include common soil
bacteria and bacteria that live in and on the body.

3. Thermophiles

Heat-loving bacteria.
Their optimum growth temperature is between 45 70C and are commonly found in hot springs and in
compost heaps.

Temperature
5. Hyperthermophiles

Bacteria that grow at very high temperatures.

Their optimum growth temperature is
between 70 - 110C.
They are usually members of the Archae and
are found growing near hydrothermal vents at
great depths in the ocean.

Moisture
Moisture is required to:
carry food in solution into the cell,
carry waste in solution away from the cell, and
maintain the moisture content of the
cytoplasm.