MEDICAL MICROBIOLOGY I Lecture 1 Laboratory Safety and Bacterial Growth
Nov 06, 2015
MEDICAL MICROBIOLOGY I
Lecture 1Lecture 1
Laboratory Safety and Bacterial
Growth
Laboratory Safety
Identify and evaluate hazards
Type of hazard:
1. Biohazards (infectious agents or items contaminated with them)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, 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 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 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 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 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 Equipment5. Gloves
prevent exposure to microorganisms
glove material is rarely completely
impermeable; it delays penetration of harmful
material for a time sufficient to provide 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) mask-
better protection better 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 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 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 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 / tiedsealed / 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: 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 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 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 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 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. 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: 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 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 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 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 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 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 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 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. Nutrition
2. Oxygen2. Oxygen
3. pH
4. Temperature
5. 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.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 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. Photoautotrophs
2. Photoheterotrophs
3. Chemoautotrophs
4. Chemoheterotrophs
Photoautotroph
Photoautotrophs use light as an energy source and carbon dioxide as their main carbon source.
They include photosynthetic bacteria (green 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 Instead they use organic compounds as a
carbon source. They include the green non-
sulfur 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: 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 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 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. 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 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 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 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.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. 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 maintain the moisture content of the
cytoplasm.