BIOLOGY & SCIENCE: Microbiology

Showing posts with label Microbiology. Show all posts

Thursday, April 30, 2009

article : Content the MICROBIOLOGY TOPICS

This relatively new science began with the erroneous belief in spontaneous generation (abiogenesis) and matured into the germ theory on which today’s understanding of infectious disease is based.

MICROBIOLOGY TOPICS

VIRTUAL MICROBIOLOGY CLASSROOM

Click here to visit the online materials used in an actual college-level Microbiology Course. The Virtual Microbiology Classroom provides access to a wide range of educational resources including Power Point Lectures, Study Guides, Review Questions and Practice Test Questions.

HISTORY OF MICROBIOLOGY

From spontaneous generation (abiogenesis) to modern immunology, this link will take you to a series of articles summarizing the key contributors and discoveries.

SPONTANEOUS GENERATION DEBATE

GERM THEORY OF DISEASE

EARLY HISTORY OF IMMUNOLOGY

CLASSIFYING ORGANISMS (SYSTEMATIC & TAXONOMY)

Linnaeus and Binomial Nomenclature

TYPES OF MICROBES

All life is composed of cells, and only two basic models exist; prokaryotes and eukaryotes. All bacteria are prokaryotic cells. Viruses are acellular, or non-living particles that are able to cause infectious disease.

PROKARYOTES (Bacteria & Archaea)

Prokaryotic Microbes
Classification of Bacteria
What Are Normal Flora? 2/10
Probiotic Bacteria that Promote Digestive Health 11/09

EUKARYOTIC MICROBES

Eukaryotic Microbes

VIRUSES

HOW TO USE A MICROSCOPE

SPO provides a number of articles on how to properly use a compound light microscope. The main page provides descriptions and links to help you easily find the specific information that you are looking for.

BACTERIAL IDENTIFICATION

Microbes are tiny and often colorless. That wouldn't be a problem if we didn't so often have to identify them. Without the ability to ID specific microbes, the treatment of infectious disease would not be possible. The following links provide information on several methods used to sleuth out the identity of different bacteria, including differential staining, specialized media and other metabolic tests.

MICROBIAL CONTROL (How to Kill Microbes)

Control of microbial growth (killing microbes) can be carried out number of ways, through use of chemical, physical and chemotherapeutic (antibiotic, antiviral and antifungal) agents.

Here are links to additional pages each covering a specific microbial control method.

Physical Control of Microbes (Heat, UV radiation)
Chemotherapeutic Control of Microbes (Antibiotics and other meds)
Chemical Control of Microbes (Disinfectants, cleaners like bleach and lysol)

INFECTIOUS DISEASE

So small that they can only be seen with a light microscope or electron microscope, humans have battled these tiny pathogens throughout our history. The following links lead to articles that describe and give examples of infectious diseases caused by eukaryotic pathogens, bacteria, viruses, viroids and prions.

Sources

Image

van Leeuwenhoek, Public Domain, Wiki

article : Bacterial Endospore Stain Protocol

Differential Test to Identify Bacteria Genera Bacillus & Clostridium

Read more: "Bacterial Endospore Stain Protocol: Differential Test to Identify Bacteria Genera Bacillus & Clostridium" - http://bacteriology.suite101.com/article.cfm/bacterial_endospore_stain#ixzz0E7UCb8xK&A

Before discussing the procedue for staining endospores, it is important to understand what these unique structures are and how the power of the endospore was first dicovered.

Heat Resistant Bacteria

John Tyndall was a 17th century Irish-born English-bred physicist who made many contributions to science, one of which was the discovery that some microbes existed in two forms:

heat-stable form (endospore)
heat-sensitive form (vegetative cell)

Tyndall found that it took either prolonged or intermittent heating to destroy the resistant heat-stable form. The outcome of this research was a method of sterilizing liquid by heating it to boiling point on successive days ("Tyndallization").

"Tyndallization" is useful for sterilization of growth media in science classes and other situations where autoclaves (instruments that use both heat and pressure to sterilize) are not available for pressure sterilization.

What Is an Endospore?

Endospores are produced by very few types of bacteria, most notably the genera Clostridium and Bacillus. These protective structures are made through a process known as sporulation in response to extreme environmental conditions, such as high temperatures, desiccation, chemicals, changes in pH and lack of food.

In the dormant, inert endospore state, bacteria do not metabolize or reproduce, but exist in a type of suspended animation, much like the seeds of plants do. When environmental conditions again become favorable, the endospore germinates, returning the bacterium to its normal active and reproducing metabolic state.

Staining Bacterial Endospores

Normal water-based techniques, such as the Gram stain, will not stain these tough, resistant structures. In order to stain endspores, malachite green must be forced into the spore with heat, in much the same way that carbol fuschsin is forced through the waxy mycolic acid layer of Mycobacterium in the Acid-fast Stain.

The protocol for differentially staining endospores and vegetative cells is as follows:

Place a strip of blotting paper over the slide.
Place the covered slide over a screened water bath and then saturate blotting paper with primary stain malachite green.
Allow the slide to sit over the steaming water bath for 5 minutes, reapplying stain if it begins to dry out.
Remove blotting paper and rinse slide with water until water runs clear.
Flood slide with the counterstain safrinin for 20 seconds and then rinse.
View specimin under oil immersion (magnification of 1000xTM) with a light microscope.

After this staining procedure, the endospores will appear green, having retained the primary stain, malachite green. The vegetative cells (bacteria are in the active, metabolizing state) will appear pink, having retained the counterstain, safrinin.

Problems Interpreting Endospore Stain

It should be noted that any debris on the slide can also take up and hold the green stain. Everything that ends up green on the slide is not necessarily an endospore. Endospores are small and typically oval. Large or irregular globs of green on the slide may be artifacts.

Acid-fast cells, such as members of Mycobacterium and Nocardia have waxy molecules in their cell wall that will take up and retain the malachite green stain when subjected to the endospore staining process. The uniformly green appearance of endospore stained Acid-fast cells doesn’t mean that they produce endospores. These are vegetative cells that have taken up color from the heat driving malachite green into their waxy cell wall.

Additional Microbiology Information

For more information on prokaryotes and cell biology, go to the excellent websites of SPO Virtual Microbiology Classroom and Microbe Wiki.

Sources

Bauman, R. (2005) Microbiology.

Park Talaro, K. (2008) Foundations in Microbiology.

The copyright of the article Bacterial Endospore Stain Protocol in Bacteriology is owned by Tami Port. Permission to republish Bacterial Endospore Stain Protocol in print or online must be granted by the author in writing.

article : Differential staining for Clostridium bacteria

The final differential stain that we will be learning in lab is called an Endospore stain 7/09. It is a special staining procedure that allows us to stain both vegetative (active) cell and endospores.

What are Endospores?

Two genera of bacteria, Clostridium and Bacillus, produce endospores. Endospores are tough, resistant structures that allow the bacteria to essentially exist in ‘suspended animation.’ Endospores do not metabolize and do not reproduce but merely exist, much like plant seeds, until exposed to environmental conditions suitable for bacterial growth.

Example of Clostridium bacteria with characteristic drumstick-shaped endospore-producing cells. The dark rod-shaped cells are vegetative, actice cells. The clear ovals are endospores, and the objects consisting of both dark rod and clear oval are vegetative cells producing endospores.

Endospore Sporulation

Endospores are made through a process known as sporulation in response to extreme environmental conditions. Extreme environments include high temperatures, drying out (dessication), extremes in pH, bacteriocidal chemicals and lack of food. When environmental conditions are favorable, the endospore germinates. Upon germination, the cell returns to its normal metabolic state, capable of reproduction.

Staining Endospores

Normal staining techniques will not stain the resistant endospores. Here are the steps that are required:

Malachite Green: The stain, malachite green, is forced into the spore with heat much like the carbol fuschsin was forced through the waxy mycolic acid layer of Mycobacterium. After flooding the slide with malachite green, it is suspended over a boiling water bath for 5 minutes and then rinsed with water.
Safrinin: Once the endospore is stained, the counter stain, Safranin, provides color for the vegetative (i.e. metabolically active) cells.

What Vegetative Cells and Endospores Look Like

Below is a photo of Bacillus bacteria that have been stained using the endospore stain. At the end of this differential staining process the vegetative cells (active, metabolizing cells) are pink and the endospores, if present, are green.

ADDITIONAL ARTICLES ABOUT ENDOSPORE STAINING

Follow the links below to articles explaining the Endospore stain procedure and the differences between vegetative cells and endospores.

Bacterial Endospore Stain Protocol: Differential Test to Identify Bacteria Genera Bacillus & Clostridium 7/09

Endospore staining involves application of a series of dyes. Malachite green stains endospores and safrinin dyes vegetative cells pink. Here's endospore stain procedure.

Differential Staining & Bacterial Controls: Positive & Negative Controls: Gram, Acid-fast & Endospore Stains 7/09

Bacterial controls are often used with differential stains as examples of typical positive and negative stain reactions; helpful references when identifying unknowns.

Sources:

Microbiology class laboratory material appearing on this website is adapted from the Applied Microbiology laboratory manual by Cynthia Schauer.

Images:

Heat fixed slide prepared for Gram staining. Positive control is on the left and negative control is on the right, T. Port.

Thursday, March 12, 2009

article : In Vitro Antibiotic Susceptibility Testing

Performed to determine the susceptibility of the organism isolated from the diseased host; Generate antibiogram(s)

Broth Dilution MIC test

Agar Dilution MIC test

Broth/Agar MBC test

Agar Diffusion (Kirby-Bauer Disk) Test: Measure diameter of zone of inhibition; Read as susceptible, intermediate, or resistant

Factors influencing zones of inhibition on agar

Concentration of bacteria spread onto agar plate
Pathogen susceptibility
Antibiotic diffusion effects
Agar depth
Growth rate
Temperature
Nutrient availability
Drug antagonists

Factors influencing diffusion of antibiotic

Concentration of antibiotic: Kirby-Bauer disks, E-tests have standardized concentrations
Molecular weight of antibiotic
Water solubility of antibiotic
pH and ionization
Binding to agar

Modern Commercial Kits (e.g., E-test strips (up to six) on an agar plate and can be read out as MIC)

article : Physiological and Biochemical Mechanisms of Drug Resistance

Bacteria may Demonstrate any of Five General Mechanisms of Antibiotic Resistance:

Lack of entry; Decreased cell permeability
Greater exit; Active efflux
Enzymatic inactivation of the antibiotic
Altered target; Modification of drug receptor site
Synthesis of resistant metabolic pathway

These Mechanisms can be Grouped into Three Broad Categories:

Permeability Mechanisms

Lack of entry; Decreased cell permeability

Greater exit; Active efflux

Enzymatic Inactivation of the Antibiotic Altered Target or Pathway

Altered target; Modification of drug receptor site

Synthesis of resistant metabolic pathway

article : Antibiotic Resistance

Bacterial Resistance to Antibiotics is either:

Intrinsic (inherent) or phenotypic
Acquired via acquisition of foreign resistance genes
Acquired via mutational events in the native genome

Intrinsic Resistance: organism is inherently not susceptible to the antibiotic

Phenotypic Resistance (non-genetic) (e.g., non-growing cells; gram-negative, outer cell membrane)

Genotypic Resistance: Exchange of r-Determinants (Genes that confer resistance to specific antimicrobial agents); Transfer and recombination of resistant mutant genes is possible through normal bacterial genetic exchange mechanisms (conjugation; transduction; transformation)

Plasmids: Multidrug (multiple) resistance is possible; Can cross species barrier and closely related strains may acquire r-determinants

Plasmid: covalently closed circular extrachromosomal DNA

Dispensable
May carry genes for drug resistance; metabolic enzymes; virulence factors (e.g., exotoxins)
Restricted or broad host range
Small size (~5 Md) are non-conjugal; Large (20-200 Md) can be conjugal

Plasmid transfer between cells

Bacterial conjugation ("sex"):Replication and transfer of the conjugal plasmid via cell-to-cell contact through an F-pilus encoded by tra (transfer) genes
Transduction: Transferred by phage
Transformation possible

Transposons (Tn) (plasmid or chromosomal): Genes transferable within a replicon via self-excision; Multidrug resistance is possible; Can cross species barrier

Integrons found on transposons or plasmids; Contains the gene and the site for incorporating resistance genes as cassettes allowing expression of the genes; Multidrug resistance is possible