The following list of some of the general areas in which the expertise of a microbiologist might be used:• medicine• environmental science• food and drink production• fundamental research• agriculture• pharmaceutical industry• genetic engineering.

‘Microbiology’

An easy word to define: the science (logos) of small (micro) life (bios), or to put it another way, the study of living things so small that they cannot be seen with the naked eye.Microorganisms are organized into six fields of study: bacteriology, virology, mycology, phycology, protozoology, and parasitology.

BACTERIOLOGYBacteriology is the study of bacteria. Bacteria are prokaryotic organisms. A prokaryotic organism is a one-celled organism that does not have a true nucleus.

Bacterial Cell

The Gram stain is a differential staining technique that can differentiate eubacteria into two distinct groups: (i) those that acquire blue-purple color at the end of the staining ; these are called gram-positive bacteria; and (ii) bacteria which acquire pink to red color and are designated gram-negative bacteria.

It is one of the most important identification criteria in any identification scheme for all types of bacterial isolates. Knowing the Gram reaction of a microorganism, in addition to morphology characterization, rules out a huge number of other bacterial.

The technique involves the use of :

A primary stain, most often crystal violet (CV), as the blue-violet dye.

A mordant, a solution of iodine that forms a complex with CV in stained bacterial cells.

A decolorizing solution, a solvent (alcohol or alcohol-acetone mixture) that removes color of CV from some bacterial types but not others.

A counterstain, most often safranin, which confers red color on decolorized cells, thus contrasting the purple color of cells that retain CV after decolorization.

Procedure for your reference:-

http://www.wisc-online.com/Objects/ViewObject.aspx?ID=MBY4808

Gram stain

Type of micro-organism based on Oxygen Requirements

1.    strict or obligate anaerobes – oxygen kills the bacteria; ex. Clostridium tetani2.    strict or obligate aerobes – lack of oxygen kills the bacteria; ex. Pserdomonas3.    facultative anaerobes – can shift their metabolism (anaerobic if oxygen is absent or aerobic if oxygen is present); ex. E. coli, Staphylococcus4.    aerotolerant – the bacteria don’t use oxygen, but oxygen doesn’t harm them; ex. Lactobacillus

Fermentor and non fermentor bacteria:

Lactose is a carbohydrate that may be used as a nutrient The utilization of lactose is importnat in identifying bacteri as fermenter and non fermenter.

Fermenter (lactose positive) means that the organism can use lactose as an energy source.

E.coli, Klebsiella pneumoniae, Enterobacter spp, Proteus mirabilis, Citrobacter spp., Salmonella spp., Appers pink on MacConkay agar.

Non Fermenter (lactose negative) means that the organism can not use lactose for energy production.

Acinetobacter baumannii, Pseudomonas spp.Appers colorless on MacConkey Agar 

Two Types of Reproduction in Microbes:

Asexual

1.) Binary Fission - Bacterial reproduction occurs through fission, a primitive form of cell division.

2.) Budding - A few bacteria and some eukaryotes (including yeasts) may also replicate by budding, forming a bubble-like growth that enlarges and separates from the parent cell.

Reference:

http://bugs.bio.usyd.edu.au/learning/resources/CAL/Microconcepts/moviePages/bacteriaDivision.html

Sexual Sex in bacteria differs somewhat from what we consider sex in eukaryotes. It involves the plasmid, which has several important characteristics:

1. A plasmid is a loop of DNA. Plasmids can multiply autonomously within the cell. Time of reproduction one or more plasmids in each cell.

2. Many plasmids can insert into the DNA of the nucleus, and detach from it. In doing so, the plasmid may leave part of the plasmid DNA behind, and take some of the nuclear DNA with it.

3. plasmids can transfer from cell to cell. The cells need not be of the same bacterial 'species'.

Reference:http://bugs.bio.usyd.edu.au/learning/resources/CAL/Microconcepts/moviePages/conjugation.html

Microbial Growth

1. Lag Phase - In the lag phase, the number of cells doesn't increase. However, considerable metabolic activity is occurring as the cells prepare to grow. 2. Log Phase (logarithmic or exponential phase) - cell numbers increase exponentially. Organisms in a tube of culture medium can maintain log growth for only a limited time, as nutrients are used up, metabolic wastes accumulate, microobes suffer from oxygen depletion.

3. Stationary Phase - The number of cells doesn't increase, but changes in cells occur: cell become smaller and synthesize components to help them survive longer periods without growing.

4. Death Phase - In this phase, cells begin to die out. Death occurs because cell have depleted intracellular ATP reserves. Not all cells necessarily die during this phase!

Laboratory Exercise

Media & Culture:

Collecting and Culturing Bacterial Samples

How is media made? • When lab personnel make

media they measure out a quantity of dry powdered nutrient media, add water and check the pH.

• They pour the media into bottles, cap it and autoclave.

• The autoclave exposes the media to high temperature (121°C) and pressure (15 psi, pound per square inch) for 20 minutes.

• Once the media is autoclaved it is considered sterile (all life forms killed).

Selective media contain ingredients that inhibit the growth of some organisms but allow others to grow.

For example, mannitol salt agar contains a high concentration of sodium chloride that inhibits the growth of most organisms but permits staphylococci to grow.

Differential media contain compounds that allow groups of microorganisms to be visually distinguished by the appearance of the colony or the surrounding media, usually on the basis of some biochemical difference between the two groups.

Blood agar is one type of differential medium, allowing bacteria to be distinguished by the type of hemolysis produced.

Differential & selective selective media

MacConkey and eosin-methylene blue agars, which are selective for gram-negative coliforms and can differentiate lactose-fermenting and non-lactose-fermenting bacteria.

Growth Factors 1. pH – bacteria can classified as:a. acidophiles (acid-loving) – grow best at a pH of 1 to 5.4; Ex. Lactobacilllus (ferments milk)b. neutrophiles – exist from pH to 5.4 to 8.5; most bacteria that cause human disease are in this category.c. alkaliphiles (base loving) – exist from pH to 7.0 to 11.5; ex. Vibrio cholerae (causes cholera) 2. Temperature – bacteria can be classified as:a. psychrophiles (cold-loving) 15oC to 20oC; some can grow at 0oC.b. mesophiles - grow best between 25oC and 40 C; human body temp is 37oC. c. thermophiles (heat-loving) – 50oC to 60oC; found in compost heaps and in boiling hot springs. 3. Moisture – only the spores of spor-forming bacteria can exist in a dormant state in a dry environment. 4. Hydrostatic pressure – pressure exerted by standing water (ex. lakes, oceans, etc.); some bacteria can only survive in high hydrostatic pressure environments (ex. ocean valleys in excess of 7000 meters); the high pressure is necessary to keep their enzymes in the proper 3-D shape; without it, the enzymes lose their shape and denature and the cell dies. 5. Tonicity (hypotonic, hypertonic, isotonic) – The use of salt as a preservative in curing meats and the use of sugar in making jellies is based on the fact that a hypertonic environment kills or inhibits microbial growth. Halophiles (salt lovers) inhabit the oceans. 6. Radiation – UV rays and gamma rays can cause mutations in DNA and even kill microorganisms. Some bacteria have enzyme systems that can repair some mutations.

TSY

Growth Media• Bacteria and other microbes have

particular requirements for growth.

• In order to successfully grow bacteria in lab, we must provide an environment suitable for growth.

• Growth media (singular = medium) are used to cultivate microbial growth.

• Media = mixtures of nutrients that the microbes need to live. Also provides a surface and the necessary moisture and pHto support microbial growth.

• Tryptic Soy Agar (TSY) is the medium that we most oftime use. Complex nutrient media which supports the growth of a wide variety of microbes.

Specialized Media:

McConkey’s, Mannitol Salt & Blood Agar

McConkey’s = lighter, purplish-pinkMannitol Salt = orangish-pinkBlood Agar = very dark red

Differential &

Selective Specialized

Media

MacConkey's (MAC)

media is both slective and differential.

1. Selective because it only grows Gram Negative bacteria. Inhibits the growth ofGram Positive bacteria.

1. Differential because neutral red (pH-sensitive

dye) and lactose (type of sugar) have been added to media.

- Bacteria that use lactose for food (lactose fermenters), produce acidic metabolites that trigger the ph sensitive dye to turn pink.

- So lactose fermenting bacteria will grow in bright pink colonies while

non-lactose fermenters will be colorless and clear.

Mannitol Salt (MSA)

is both selective & differential.

1. Selective because it has a high NaCl (7.5%) concentration, and few types of bacteria can grow on this hypertonic medium.

Members of genus Staphylococcus grow well on this media.

2. Differential because this medium contains a pH-sensitive dye to identify organisms that ferment mannitol.

Organic acids wastes mannitol fermenters produce change the medium from red to yellow.

MSA works well for identifying pathogenic staphylococci, such as Staphylococcus aureus, which will ferment mannitol.

Most non-pathogenic staphylococci (Staphylococcus epidermidis) will not ferment mannitol.

Blood agar (BAP)

Most specimens received in a clinical microbiology lab are plated onto Blood Agar. It is an enriched medium that will grow even fastidious bacteria.

Also contains 5% sheep blood.

This media is not selective. It is enriched and differential:

Certain bacteria produce enzymes (hemolysins…say hemo-lice-ins) that act on red cells to produce either:

* Beta hemolysis: Enzymes lyse the blood cells completely, producing a clear area around the colony.

* Alpha hemolysis: Incomplete hemolysis produces a greenish discoloration around the colony.

* Gamma hemolysis: No effect on the red cells.

Microbiologist are trying to detect Group A beta hemolytic Streptococcus pyogenes (a Gram-positive cocci-shaped bacteria that causes Beta hemolysis on blood agar.)

Normal flora of the throat will exhibit alpha or gamma hemolysis.

Tryptic soy agar (TSA) Type: General Purpose: Cultivation of nonfastidious bacteria

Interpretation: Growth indicates nonfastidious bacteria present

Tryptic soy agar - Pseudomonas aeruginosa

Note the blue-green color due to pyocin production by the bacteria.

Tryptic soy agar - Staphylococcus aureus

Note the carotenoid pigment typical of S. aureus.

Tryptic soy agar, uninoculated

Chocolate agar

Type: Enriched Purpose: Cultivation of fastidious organisms such as Neisseria or Haemophilussp. Interpretation: Some organisms grow on chocolate agar that do not grow on standard media.

These bacteria need growth factors, like NAD (factor V) and hemin (factor X), which are inside red blood cells; thus, a prerequisite to growth is lysis of the red blood cells. The heat also inactivates enzymes which could otherwise degrade NAD.

Chocolate agar - Staphylococcus aureus

Note: luxuriant growth with yellow pigmented colonies.

Type: Selective and differential, Purpose: Contains bile salts and crystal violet which selects for gram-negative enterics, differentiates lactose fermenters from nonfermenters. Selective because it only grows Gram Negative bacteria. Inhibits the growth ofGram Positive bacteria.

Differential because neutral red (pH-sensitive dye) and lactose (type of sugar) have been added to media.

- Bacteria that use lactose for food (lactose fermenters), produce acidic metabolites that trigger the ph sensitive dye to turn pink.

- So lactose fermenting bacteria will grow in bright pink colonies while non-lactose fermenters will be colorless and clear.

MacConkey agar

MacConkey agar - Escherichia coli

Note: red colonies and red precipitate due to acid production as a result of lactose fermentation.

MacConkey agar - Salmonella enteritidis

Note: growth, but no fermentation of lactose. Colorless colonies, medium is slightly yellow due to the increased pH resulting from bacterial digestion of peptone in the medium.

Measuring Numbers of Microbes A.  Indirect Measurements (measure a property of the mass of cells and then ESTIMATE the number of microbes.

 1.    Turbidity – Can hold tube up to the light and look for cloudiness as evidence of growth (difficult to detect slight growth).  the greater the mass of cells in the culture, the greater its turbidity (cloudiness) and the less light that will be transmitted. 

2.    Metabolic Activity  - 3 ways:a.       The rate of formation of metabolic products, such as gases or acids, that a culture produces.b.      The rate of utilization of a substrate, such as oxygen or glucose.c.       The rate of reduction of certain dyes.  Ex. methylene blue becomes colorless when reduced.

B.  Direct Measurements - Give more accurate measurements of numbers of microbes.

1.    Direct Counts - Coulter Counter - electronic counter; rapid & accurate only if bacterial cells are the only particles present in the solution.

2.    Plate Count – Bacterial colonies are viewed through the magnifying glass against a colony-counting grid.

3.    Filtration - A known volume of liquid or air is drawn through a membrane filter by vacuum.  The pores in the filter are too small for microbial cells to pass through.  Then the filter is placed on an appropriate solid medium and incubated.  The number of colonies that develop is the number of viable microbial cell in the volume of liquid that was filtered. This technique is great for concentrating a sample, ex. a swimming pool, where small populations may go undetected using some other methods.

TYPES OF INFECTIOUS DISEASE   Acute disease – develops rapidly and runs its course quickly            Chronic disease – develops more slowly, is usually less severe, and persists for a long period.            Latent disease – characterized by period of inactivity  (ex. Herpes)            Local infection – confined to a specific area            Systemic infection – generalized infection; affects most of the body.             Septicemia – pathogens are present in and multiply in the blood            Primary infection – initial infection in a previously healthy person.            Secondary infection – follows a primary infection (ex. a bacterial infection following a cold). Mixed infection  - caused by several species of organisms present at the same time.

             STAGES OF INFECTIOUS DISEASE    A.   INCUBATION PERIOD – Time between infection and the appearance of signs and symptoms.  Although the infected person is not aware of the presence of an infectious agent, person can spread the disease to others.  Each infectious disease has a typical incubation period. B.    PRODROMAL PHASE (prodromos = forerunner) - Short period during which nonspecific, often mild, symptoms such as muscular pain, headache sometimes appear.  You feel like you’re coming down with something. C.    INVASIVE PHASE – Period during which the individual experiences the typical signs and symptoms of the disease (fever, nausea, rash, cough, etc.).  During the acme part of this phase, signs and symptoms reach their greatest intensity.  In some diseases this phase may be fulminating (sudden and severe), in others it may be persistent or chronic.  A period of chills followed by fever marks the acme of many diseases.  The battle between pathogens and host defenses is at its height during this stage.   D.   DECLINE PHASE – Symptoms begin to subside as the host defenses and the effects of treatment if being administered finally overcome the pathogen.   E.    CONVALESCENCE PERIOD – Tissues are repaired, healing takes place, and the body regains strength and recovers.  Individuals no longer have disease symptoms, but they may still be able to transmit pathogens to others.

MECHANISMS OF ANTIMICROBIALRESISTANCE

There are a number of ways by which microorganisms are resistant to antimicrobial agents.

These include:

1) the bacteria produce enzymes that either destroy the antimicrobial agent before it reaches its target or modify the drug so that it no longer is recognized by the target.

2) the cell wall becomes impermeable to the antimicrobial Agent

3) the target site is altered by mutation so that it no longer binds the antimicrobial Agent

4) the bacteria possess an efflux pump that expels the antimicrobial agent from the cell.

5) specific metabolic pathways in the bacteria are genetically altered so that the antimicrobial agent cannot exert an effect.

Production of Enzymes

• Beta-lactamases are enzymes that hydrolyze beta-lactam drugs. As a result the cell is resistant to the action of the beta lactam drugs.

– In gram-negative bacteria the beta lactam drugs enter the cell through the

porin channels and encounter beta-lactamases in the periplasmic space.

The beta-lactamases destroy the beta-lactam molecules before they have a

chance to reach their PBP targets.

– In gram-positive bacteria the beta-lactamases are secreted extracellularly

into the surrounding medium and destroy the beta-lactam molecules before

they have a chance to enter the cell.

• Aminoglycoside-modifying enzymes: Gram-negative bacteria may produce

adenylating, phosphorylating or acetylating enzymes that modify anaminoglycoside so that it is no longer active.

• Chloramphenicol acetyl transferase: Gram-negative bacteria may produce an acetyl transferase that modifies chloramphenicol so that it is no longer active.

Bacterial Outer Membrane Impermeability

• Alteration of porins in gram-negative bacteria:

– Gram-negative bacteria may become resistant to beta lactam antibiotics by developing permeability barriers. This usually is caused by altered porin channels in the outer membrane that no longer allow the entrance and passage of antibiotic molecules into the cell. When beta-lactams cannot reach the PBPs, the cell is resistant.

Alteration of Targets

1. PBPs in both gram-positive and gram-negative bacteria may be altered through mutation so that beta lactams can no longer bind to them; thus the cell is resistant to these antibiotics.

2. Ribosomes. Methylation of ribosomal RNA confers macrolide resistance.

3. DNA gyrase and topoisomerase IV. Mutations in the chromosomal genes for

DNA gyrase and topoisomerase IV confer quinolone resistance.

INTRINSIC VS. ACQUIRED RESISTANCE

Resistance is an intrinsic or innate property.

This intrinsic resistance may be due to one or more of the resistance mechanisms previously described. For example, E. coli is intrinsically resistant to vancomycin because vancomycin is too large to pass though porin channels in their outer membrane. Gram-positive bacteria, on the other hand, do not possess an outer membrane and thus are not intrinsically resistant to vancomycin.

Bacteria also can acquire resistance to antimicrobial agents by genetic events such as mutation, conjugation, transformation, transduction and transposition.

• Mutation. Mutation occurs at a relatively low frequency but, when the bacteria are exposed to the antibiotic, only the mutant cell survives. It then multiplies and gives rise to a resistant population. Spontaneous mutations may also occur in plasmids. For example, mutations in plasmids containing genes for beta-lactamase enzymes can result in altered beta-lactamases often with extended activity.

• Conjugation. Bacteria often contain extrachromosomal genetic elements called plasmids, many of which carry genes for antimicrobial resistance. When two bacterial cells are in close proximity, a bridge-like structure known as a pilus forms between them. This allows a copy of the plasmid as it is replicated, to be transferred to another cell.

• Transformation. Bacteria may encounter fragments of DNA that carry antimicrobial resistance genes. These fragments are taken into the cell by a process called transformation. The DNA fragment is incorporated into the host cell chromosome by recombination and the resulting cell is resistant.

• Transposition. Specialized genetic sequences known as transposons are “mobile” sequences that have the capability of moving from one area of the bacterial chromosome to another or between the chromosome and plasmid . Since transposon DNA can carry genes for antimicrobial resistance they have contributed to the development of plasmids encoding genes for antibiotic resistance. Some transposons are capable of moving from one bacterium to another without becoming incorporated into a chromosome or plasmid.

Transformation

Contaminant vs Pathogen

Respiratory Tract

StreptococciStreptococcus pneumoniaeS. aureus (predominant)H. influenzaeNeisseria meningitidisEnterobacteriaceae (predominant)Pseudomonas (predominant)Nocardia sppMoraxella catarrhalis (predominant)

Gastrointestinal Tract

Salmonella sppShigella sppCampylobacter jejuniAeromonas/PlesiomonasYersinia enterocoliticaVibrio sppS. aureus (in the context of food poisoning)

UrineEnterobacteriaceaeEnterococcus sppPseudomonas sppstreptococci (in pregnancy)S. aureus