How micro-organism function (microbial physiology)

Mr. O.Brown

Introduction

• In this unit, we be focusing on population of microbial cells and how their numbers increase through growth and decrease or disappear as a result of death.

Reproduction

• Microbial growth – an increase in a population of microbes rather than an increase in size of an individual

• Result of microbial growth is discrete colony – an aggregation of cells arising from single parent cell

• Reproduction results in growth

The Way Microorganisms Grow

• Most Bacteria elongate and divide by binary fission (cleavage near midpoint to form two daughter cells of approximately equal size).

• division exactly in half• most common means of bacterial

reproduction– forming two equal size progeny– genetically identical offspring– cells divide in a geometric

progression doubling cell number

Cont’d

• Some unicellular microorganisms, including a few bacteria a few bacteria, replicate by budding (forming a bubblelike growth that enlarges and separates from the parent cell).

Phases of Growth

• Exponential growth can not continue for long. The increasing number of cells uses nutrients and produces waste at ever increase rates.

• Usually, essential nutrients runs out. Sometimes toxic products stop further growth. Whatever the cause, growth rate slows and eventually stops.

• Then the culture is said to pass from the exponential phase (also called the logarithmic or log phase) of growth to the stationary phase.

Phases of Growth

Stages in the normal growth curve

1. lag phase – “flat” period of adjustment, enlargement; little growth

2. exponential or log phase – a period of maximum growth will continue as long as cells have adequate nutrients and a favorable environment

3. stationary phase – rate of cell growth equals rate of cell death caused by depleted nutrients and O2, excretion of organic acids and pollutants

4. death phase – as limiting factors intensify, cells die exponentially in their own wastes

Factors Affecting the Growth of Microorganisms

• There are several factors that affect the growth of microorganisms, these are:

• Nutrients• Temperature• pH levels• Moisture • Oxygen and • Osmotic Potential

Nutrients• All microorganisms need food. The food sources can vary,

but the organisms primarily extract carbon and nitrogen from substances such as proteins, fats and carbohydrates. Some microorganisms seek out and absorb such particles.

• Others may perform chemical reactions with surrounding elements such as carbon dioxide to gain what they need, while still others can produce their own simple sugars through photosynthesis similar to plants.

• Nitrogen, which is used to synthesize proteins, can be taken from the surrounding atmosphere or from other organic matter.

Temperature• In general, the higher the temperature, the more

easily microorganisms can grow up to a certain point. Very high and very low temperatures both obstruct the enzyme processes microorganisms depend on to survive, but individual species of microorganisms have grown to prefer different levels of temperature.

• Scientists usually divide them into three different groups: psychrophiles, mesophiles and thermophiles. Psychrophiles prefer temperatures from 0 to 5 degrees Celsius; mesophiles like it in the middle, 20-45 degrees Celsius; and thermophiles like it hot, thriving in temperatures around or above 55 degrees.

Note the drop-off as you get closer to the Optimum!

Food Spoilage Temperatures

pH Levels• Microorganisms also prefer a certain pH level in

the substance or environment in which they grow--that is, they prefer to have particular acidic qualities in their surroundings.

• Most microorganisms, including most human pathogens, are neutriphils, organisms that prefer a neutral pH level.

• Some like high pH levels, but most often, if conditions are too acidic, then the organism's enzymes break down.

Cont’d

• Acidophilies have their growth optimum between pH 0-5.5; neutrophiles between pH 5.5-8.0; and alkaliphiles (alkalophiles), between pH 8.0 – 11.5.

pH• Most bacteria prefer neutral pH (6.5-7.5)• Molds and yeast grow in wider pH range, but

prefer pH between 5 and 6.• Acidity inhibits most microbial growth and is

used frequently for food preservation (e.g. pickling).

• Alkalinity inhibits microbial growth, but not commonly used for food preservation.

• Low pH also increases the effectiveness of heat treatments. Acidic foods such as tomatoes and fruits can be canned safely merely by boiling.

Moisture• The free flow of water is vital to microorganisms

for their cells to exchange materials and for their metabolic processes.

• All microorganisms require some level of water, but a few can survive in low-moisture conditions by conserving all the water they find and by staying in a moisture-rich environment.

• As a general rule, though, the more moisture, the more microorganisms there will be found.

Oxygen

• In addition to water, microorganisms usually require the presence of certain elements in the air--gases that they absorb to produce needed nutrients. Nitrogen is one necessary element, as is oxygen.

• There are many microorganisms that require an oxygen-rich environment to survive (aerobe), but others actually flourish in low-oxygen surroundings (anaerobe). Between these two extremes is a wide variety that may prefer more or less oxygen and that will be able to flourish equally no matter how much oxygen is present.

• Almost all multicellular organisms are completely dependent on atmospheric oxygen for growth – that is, they are obligate aerobes.

Cont’d

• Microaerophiles are damaged by the normal atmospheric level of oxygen (20%) and require oxygen levels in the range of 2 to 10% for growth.

• Facultative anaerobes do not require oxygen for growth but grow better in its presence.

• Aerotolerant anaerobes grow equally well whether oxygen is present or not. In contrast, strict or obligate anaerobes are usually kiled in the presence of oxygen.

Osmotic Potential

• This is the concentration of solutes in the environment. Because a selectively permeable membrane separates microorganisms from their environment, they can be affected by changes in the osmotic concentration.

• If the solute potential of the environment is greater the water will move out the cell and the cell will die (hypertonic). This will cause the cell membrane to shrink. Used to control spoilage and microbial growth. Sugar in jelly and salt on meat.

• If the reverse occurs, water will enter the cell and cause it to burst (hypotonic).

Measuring Growth

• Viable Cell Count – Counting viable cells depends on the live cell’s ability to grow and to form a colony or develop into a turbid culture.

• Plate counts and filtration counts depend on the colony formation.

• Most probable number counts depends on the development into a turbid culture.

• The above mentioned counts are examples of viable cell count methods.

Measuring Growth

• Direct Methods of Measurement• 1. Plate Count: Most frequently used method of measuring bacterial

populations Inoculate plate with a sample and count number of

coloniesAssumptions:

- Each colony originates from a single bacterial count.- Original inoculum is homogenous- No cell aggregates are present

Measuring Microbial Growth

• Advantages:- Measure viable cells (live cell)

• Disadvantages:– Takes 24 hours or more for visible colonies to

appear.– Only counts between 25 and 250 colonies are

accurate.– Must perform serial dilution to get appropriate

numbers/plate.

Serial Dilutions are used with the Plate Count Method to Measure Numbers of

Bacteria

Measuring Microbial Growth

• Plate Count (continued):• A. Pour plate:– Introduce a 1.0 or 0.1 ml inoculum into an empty

Petri dish.– Add liquid nutrient medium kept at 50oC.– Gently mix, allow to solidify, and incubate.Disadvantages:- Not useful for heat sensitive organisms- Colonies appear under agar surface.

Measuring Microbial Growth

• B. Spread Plate:– Introduce a 0.1 ml inoculum onto the surface of

Petri dish.– Spread with a sterile glass rod.Advantages: Colonies will be on surface and not exposed to melted agar.

Pour Plates versus Spread Plates

Measuring Microbial Growth• Direct Methods of Measurement• 2. Filtration:– Used to measure small quantities of bacteria.– Example: fecal bacteria in a lake or in ocean water.– A measured volume (usually 1 to 100 ml) of

sample is filtered through a membrane filter (typically with a 0.45 μm pore size)

– The filter is placed on a nutrient agar medium and incubated

– Colonies grow on the filter instead of plates and can be counted

Measuring Microbial Growth

• Direct Methods of Measurement• 3. Most Probable Number (MPN):– Used mainly to measure bacteria that will grow on a

solid medium.– Dilute a sample repeatedly and inoculate several

broth tubes for each dilution point.– Count the number of positive tubes (cloudy) in each

set.– Statistical Method: Determines 95% probability that

a bacterial population falls within a certain range.

Measuring Microbial Growth

• 4. Direct Microscopic Count Using a Petroff Hauser:– A specific volume of a bacterial suspension (0.01 ml) is placed on a

microscope slide with a special grid.– Stain is added to visualize bacteria.– Cells are counted and multiplied by a factor to obtain

concentration.Advantages:- No incubation time requiredDisadvantages:- Cannot always distinguish between live and dead bacteria.- Motile bacteria are difficult to count.- Requires a high Concentration of bacteria (10 million/ml)

Example

• 1 ml or 1 cm3 of a sample was placed on microscope slide with a special grid and the chamber calibrated to 0.01 ml. We then view the sample on the grid and count the number cells present in the 0.01 ml to calculate the amount present in 1 ml.

• If 12 cells were counted on the grid calibrated at 0.01ml, how much cell would be in 1 ml?

Measuring Microbial Growth• Indirect Methods of Measurement• 1. Turbidity:

– AS bacteria multiply in media, it becomes turbid (cloudy).– Based on the diffraction or “scattering” of light by bacteria in a broth

culture– Light scattering is measured as optical absorbance in a

spectrophotometer– Optical absorbance is directly proportional to the concentration of

bacteria in the suspension

Advantages:- No incubation time required.Disadvantages:- Cannot distinguish between live and dead bacteria- Requires a high concentration of bacteria (10 – 100 million cells/ml).

Cont’d

• So basically, we look at how cloudy the medium is by passing light through the medium and measure the light before and after with goes through the medium.

• The more microorganisms present the more light will be absorbed, turbidity will increase, because less light is transmitted through the culture, and the reading on the spectrophotometer is higher.

Turbidity

Measuring Microbial Growth

• 2. Metabolic Activity: - As bacteria multiply in media, they produce certain products, such as:

- Carbon dioxide (gas)- Acids

- Measure the rate of formation of metabolic products.- Expensive

Measuring Microbial Growth• 3. Mass determination :

- Cells are removed from a broth culture by centrifugation (using centrifuge to rotate vessels called centrifuge tubes at a high rate of speed, generating a centrifugal force that pushes small particles, including microbial cells, to the bottom of

the tube) and weighed to determine the “wet mass.”

- The cells can be dried out and weighed to determine the “dry mass.”

- Doesn’t distinguish live and dead cells.

Control of Microorganism• 1. Sterilization:- is the process by which all living cells are

destroyed or removed from an object or habitat.

- A sterile object is totally free of viable microorganisms, spores and other infectious agents. When sterilization is achieved by a chemical agent, the chemical is called sterilant.

Cont’d

• Heat sterilization can be dry or moist. Both kill by destroying proteins.

• But dry heat kills largely by speeding up oxidations, which inactivate proteins.

• Moist heat on the other hand, denatures (alter) proteins by breaking the bonds that confer secondary and higher protein structure.

Cont’d• A. Dry Heat• In microbiological laboratory, dry-heat sterilization is

commonly used in the form of flaming and hot-air ovens.

• Direct flaming is the process of heating an instrument until its get red hot or running it quickly through a flame (depending on the material of the instrument).

• A hot-air oven may be used were severe treatments – 171oC for 1 hour, 160oC for 2 hours, or 121oC for 16 hours. Example sterilization of empty glass instruments.

Cont’d• B. Moist Heat Sterilization• Moist heat in the microbiological lab is applied either

by boiling or in autoclaves.• Moist heat is effective at lower temperatures than dry

heat, and it penetrates more quickly.• Boiling water kills most bacterial and fungal cells and

inactivates many viruses in just a few minutes. Endospores will survive boiling

• Autoclave is more effective than boiling because it uses pressure to raise the temperature considerably above that of boiling water. At a pressure of 15 pounds per square inch, the temperature in an autoclave reaches temperature of 121oC (endospores are killed).

Control of Microorganism• 2. Disinfection • - Disinfection is the killing, inhibition, or removal

of microorganisms that may cause disease; therefore disinfection is a treatment that reduces the number of pathogens to the level at which they pose no danger of disease.

• Disinfectants are agents, usually chemical, used to carry out disinfection and normally used only on inanimate (non-living) objects. A disinfectant does not necessarily sterilize an object because viable spores and few microorganisms may remain.

Control of Microorganism

• 3. Sanitization• This is closely related to disinfection.• In sanitization, the microbial population is

reduced to levels that are considered safe by health standards.

• The inanimate object is usually cleaned as well as partially disinfected. For example, sanitizers are used to clean eating utensils in restaurants.

Control of Microorganism• 4. Antiseptics• It also is frequently necessary to control

microorganisms on or in living tissue with chemical agents.

• Antisepsis is the destruction or inhibition of microorganisms on a living tissue; it is the prevention of infection or sepsis.

• Antiseptics are chemical agents applied to tissue to prevent infection by killing or inhibiting pathogen growth; they also reduce the total microbial population.

Control of Microorganism• 5. Chemotherapy• Is the use of chemical agents to kill or inhibit

the growth of microorganisms within host tissue.

Control of Microorganism• 6. Filtration• Microorganisms other than viruses can be removed

from liquids by filtration. Thus filtration does not, in a strict sense, sterilize.

• But it is used to remove cellular microorganisms from certain media, vitamin solutions, and antibiotics that are heat sensitive.

• Rather than directly destroying contaminating microorganisms, the filter simply removes them.

• There are two types of filters, these are; depth and membrane filters (read up on both).

Control of Microorganism

• 7. Radiation