Methods of classifying and identifying of microorganisms (i)

Phenotypic characteristics
• Morphology
• Staining
• Atmospheric requirements
• Biochemical and metabolic activities test
• Serology

Genotypic characteristics
• DNA fingerprinting
• Ribosomal RNA sequencing
• Amino acid sequencing
• Protein pattern analysis

Phenotype Morphology

Morphology on culture media
The characteristics of bacteria can be seen by the size, colour, shape and consistency of a colony. In a liquid medium, the region in which the organism grows depends on the oxygen requirement of that specific species.

Shapes







Bacillus (singular);
Bacilli (plural);
Rod-like shaped






Sources:
http://www.lima.ohio-state.edu/academics/biology/biodiv/monera/bacgrpos.jpg
http://www.lima.ohio-state.edu/biology/biodiv/webbact.htm



Spirilla
(Helical shape like corkscrew)












Sources:
http://www.lima.ohio-state.edu/academics/biology/biodiv/monera/spirillum.jpg



Coccus (singular);
Cocci (plural);
Spherical









Sources:
http://biology.northwestcollege.edu/biology/b1010lab/bactypes.htm



Vibrios
(Curve rods)











Sources:
http://microbewiki.kenyon.edu/images/5/55/96524K.jpg

Arrangements

Sources:
http://www.mansfield.ohio-state.edu/~sabedon/biol2010.htm














Sources:
http://www.mansfield.ohio-state.edu/~sabedon/biol2010.htm

Morphology on solid media
Pigmentation
• Transparent
• Opaque
• Colour pigment
• Fluorescent pigment

Surface properties
• Flat
• Raised
• Wrinkled
• Smooth
• Growth pattern – motile or non motile
• Filamentous and rhizoid
• Contoured













Sources: http://www.mansfield.ohio-state.edu/~sabedon/biol2010.htm

Smell
• Sweet
• Alcohol
• Foul

Morphology in broth media (test tube)
• Turbid - at the middle
• Pellicle – thick growth at the top of the tube
• Sediment – settle at the bottom

Sources: http://student.ccbcmd.edu/courses/bio141/labmanua/lab2/brothbs.html



Morphology on slant media
• Even (it follows the line of the original streak)
• Irregular (slight spreading from the original line)
• Spreading (the organisms cover the entire surfaces of the slant



















Sources: http://chsweb.lr.k12.nj.us/psidelsky/Saurs.gif

Phenotype Staining

Simple staining
It is a simple stain which uses methylene blue, crystal violet or safranin. Simple staining is sufficient determine the bacterial shape and the arrangement characteristics. An air-dried smear is stained, rinsed, dried and examined using oil immersion lens of the microscope.

Differential staining
Gram stain
• Gram positive bacteria have thick cell walls which have low lipid content. This result the cell wall to becomes less permeable during the treatment with decolourizer.
• Gram negative bacteria have thin cell walls which have higher lipid content. This result the cell wall to be soluble and thus dissolves in decolourizer.


Procedures for gram staining
Fixation-----> crystal violet-----> iodine treatment-----> decolourization----->counter stain safranin

1. Heat fix the smear
2. Cover the smear with crystal violet and rinse
3. Cover the smear with iodine and rinse
4. Apply alcohol decolourizer
5. Cover the smear with safranin and rinse

It is used for Mycobacteria because they have a thick and waxy coat. Acid-fast stain uses dyes (carbon fuchsin) with heat to allow the dye to go into the bacterium. Once acid-fast bacteria are stained, they are resistant to decolourization. This is because of its thick and waxy coat.
When non acid-fast bacteria are decolourized, they take up the counterstain, which is the methylene blue.

Result: Acid-fast – pink
Non Acid-fast - blue


Special staining
Capsular stain
Capsules do not have the same affinity for dyes as other cell components. The result of negative staining is the capsule will halo around the cell against a dark background.

Endospore stain
Endospore has a special resistant. Dormant structure formed within a cell that protects the bacteria during adverse environmental conditions.

Flagella stain
The flagella are too small to be seen under microscope. In order to see it, we have to increase the thickness of flagella by piling the stain on it.

References:
http://www.ruf.rice.edu/~bioslabs/studies/invertebrates/kingdoms.html
http://biology.northwestcollege.edu/biology/b1010lab/bactypes.htm
http://www.lima.ohio-state.edu/biology/biodiv/webbact.htm

Phenotype Agar Plate

Extracted From:
http://sg.images.search.yahoo.com/search/images?p=agar+plates&fr=yfp-t-img&ei=utf-8&js=1&x=wrt

Atmospheric Requirements
Bacterial requirements for growth include sources of energy,”organic" carbon (e.g. sugars and fatty acids) and metal ions (e.g. iron). Optimal temperature, pH and the need (or lacks of need for oxygen) are important.

A bacterial isolate can be classified into one of the five major groups :

Obligate aerobe
• An aerobic organism that requires oxygen for cellular respiration, as well as free molecular oxygen in its surroundings for growth.
• Require an atmosphere that contains 20%-21% of oxygen.
• Examples: Mycobacterium tuberculosis (acid-fast) and Bacillus (Gram-positive).

Microaerophilic aerobe
• Microaerophilic bacteria grow well in low concentrations of oxygen, but are killed by higher concentrations.
• Require an atmosphere that contains less than 5% of oxygen.
• Example: Neisseria gonorrhoeae

Facultative anaerobe
• Facultative anaerobes can perform both fermentation and aerobic respiration. In the presence of oxygen, anaerobic respiration is generally shut down and these organisms respire aerobically.
• Capable of surviving in either presence or absence of oxygen.
• Example: family of Enterobactericeae, streptococci and staphylococci.

Aerotolerant anaerobes
• Aerotolerant anaerobes are bacteria that respire anaerobically, but can survive in the presence of oxygen.
• Grows better in the absence of oxygen but can survive in atmosphere containing air and a carbon dioxide incubator.

Obligate anaerobes
• Obligate anaerobes do not carry out oxidative phosphorylation. Furthermore, they are killed by oxygen; they lack certain enzymes which detoxify both H2O2and oxygen free radicals produced as side-products during metabolism in the presence of oxygen.
• Grow in the absence of oxygen.

Nutrient Requirements
These include sources of organic carbon, nitrogen, phosphorus, sulphur and metal ions including iron. Bacteria secrete small molecules that bind iron. Siderophores are then internalized via receptors by the bacterial cell. The human host also has iron transport proteins. Thus bacteria that ineffectively compete with the host for iron are poor pathogens.

Temperature Requirements
Bacteria may grow at a variety of temperatures from close to freezing to near to the boiling point of water. Those that grow best at the middle of this range are referred to as mesophiles; which includes all human pathogens and opportunists.

pH Requirements
Many bacteria grow best at neutral pH; however certain bacteria can survive and even grow in quite acid or alkaline conditions.


Biochemical and Metabolic Activities Test
Enzymatic activities
• Enzymes are biological catalysts, or chemicals that speed up the rate of reaction between substances without themselves being consumed in the reaction
• Enzymes can accelerate, often by several orders of magnitude, reactions that under the mild conditions of cellular concentrations, temperature, pH, and pressure would proceed imperceptibly in the absence of the enzyme.
• Widely used to differentiate bacteria.

Oxidase Test
• The oxidase test is a key test to differentiate between the families of Pseudomonadaceae (ox +) and Enterobacteriaceae (ox -)
• The enzyme cytochrome oxidase is involved with the reduction of oxygen at the end of the electron transport chain. The colourless reagent used in the test will detect the presence of the enzyme oxidase and, reacting with oxygen, turn a colour.
• Positive Reaction = Purple Colour

Picture Extracted From: http://www.mc.maricopa.edu/~johnson/labtools/Dbiochem/oxi.html

Ability to utilize carbon compound
• Ability to ferment/utilize selected carbohydrate
• An example could be the ability to use glucose or citric acid as carbon source
　

Characteristics of waste or breakdown products
• In any given environment, certain bacteria are characterized by their product of metabolic activities.
• Examples: Products of CO2, H2S, O2, acid or methane.
　

Differences in metabolic pathways

• Both autotrophs and heterotrophs must break down carbon compounds to release the energy they contain to sustain their own metabolism. This breakdown process is called respiration and it occurs at all times in all living cells of all organisms, plants, animals and bacteria. Fungi and bacteria break down the carbon compounds in dead animals and plants and convert the carbon to carbon dioxide if oxygen is present, or methane if oxygen not present.

Special Media
Selective Media
Selective media are used for the growth of only select microorganisms. For example, if a microorganism is resistant to a certain antibiotic, then that antibiotic can be added to the medium in order to prevent other cells, which do not possess the resistance, from growing.


Differential Media
Differential media or indicator media distinguish one microorganism type from another growing on the same media. This type of media uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients or indicators added to the medium to visibly indicate the defining characteristics of a microorganism.
　

　
Mannitol Salt Agar
Mannitol Salt Agar (MSA):

• as a selective medium.
• A high concentration (7.5%-10%) of salt NaCl
• It is selective for members of the family Micrococcaceae and Staphylococcus since this level of NaCl is inhibitory to most other bacteria.


• as a differential medium.
• Acid production as a result of mannitol fermentation. Mannitol fermenters produce a yellow colony while non-mannitol fermenters will produce a reddish/purple colony.
• Sodium chloride is the inhibitory agent.
• Phenol red is the pH indicator.

Staphylococcus aureus and Serratia marcescens

• S. epidermidis and S. aureus on selective/differential Mannitol Salt Agar
• The Staphylococcus aureus ferments mannitol and turns the medium yellow.
• The Serratia marcescens does not grow because of the high salt content.

Picture Extracted From: http://faculty.mc3.edu/jearl/ML/ml-8.htm
http://www.austincc.edu/microbugz/mannitol_salt_agar.html


MacConkey Agar
MacConkey Agar:

• as a selective medium.
• Distinguish lactose-fermenting Gram-negative organisms from non-fermenters.
• Crystal violet, bile salts and neutral red are inhibitory agents.
• Inhibit most Gram-positive bacteria, except Enterococcus and some species of Staphylococcus.

　
• as a differential medium.
• Neutral red is the pH indicator.
• Non-Lactose fermenting bacteria use peptone instead. This forms ammonia, which raises the pH of the agar, and leads to the formation of white/colourless colonies.
• Lactose fermenting bacteria produce acid, which lowers the pH of the agar below 6.8 and results in the appearance of red/pink colonies.

Klebsiella pneumoniae ferments lactose and - E. coli and Proteus on selective /differential produces pink colonies on MAC. Micrococcus luteus does not grow in the pre presence of bile salts and crystal violet.

Picture Extracted From: http://www.austincc.edu/microbugz/html/macconkey_agar.html



Nutrient Agar
It is a growth medium used to culture microorganisms. Selective growth compounds may also be added to the media, such as antibiotics.

• Contains 0.5% gelysate peptone, 0.3% beef extract, and 1.5% agar, and will support the growth of many organisms which are not nutritionally fastidious.

• Individual microorganisms placed on the plate will grow into individual colonies.

Control of a Nutient Agar: http://zoology.okstate.edu/zoo_lrc/biol1114/study_guides/labs/lab9.htm


Streak Isolation on Nutrient: http://faculty.mc3.edu/jearl/ML/ml-8.htm


Agar Colonies on Agar: http://www.usm.maine.edu/toxicology/research/uranium.php

Blood Agar
• Consists of a basal medium such as TSA enriched with 5% sheep blood or in some locations, horse blood. This is the most commonly used medium, and supports the growth of most of the common fastidious organisms, as well as, all of the less fastidious organisms.
• Blood agar is not selective as almost any type of microbe can grow on it.
• It identifies different bacteria based on their pattern of hemolysis.

Alpha hemolysis (α-hemolysis) is present if there is a greenish darkening of the agar under the colonies. Alpha hemolysis is generally caused by peroxides produced by the bacterium. Streptococcus pneumoniae and Streptococcus viridans display alpha hemolysis.


http://telem.openu.ac.il/courses/2007a/c20237/gifs/gallery/alpha_hemolysis.htm


http://www1.indstate.edu/thcme/micro/hemolys.html



Beta hemolysis (β-hemolysis) indicates a zone of clearing in the blood agar in the area surrounding a bacterial colony. It is caused by a complete lysis of the red cells in the media. The area around and under the colonies are lightened and transparent. Streptococcus pyogenes as well as some strains of Staphylococcus aureus display beta hemolysis.


http://www.as.ysu.edu/~crcooper/3702Laboratory(F04).html


http://www1.indstate.edu/thcme/micro/hemolys.html

Gamma-hemolysis is actually a lack of hemolysis in the area surrounding a bacterial colony growing on blood agar. In fact, culture of bacteria on blood agar for the purpose of hemolysis classification is performed at 37oC in the presence of 5% CO2. This results in an overall brownish discoloration of the blood agar, from its original blood-red hue.


http://sg.images.search.yahoo.com/search/images?p=Gamma+hemolysis+&fr=yfp-t-img&ei=utf-


http://www.jlindquist.net/generalmicro/dfhemo.html

Serology Testing
• It is the scientific study of blood serum.
• Usually refers to the diagnostic identification of antibodies in the serum.
• May be performed for diagnostic purposes when an infection is suspected, in rheumatic illnesses, and in many other situations, such as checking an individual's blood type.
• Help to diagnose patients with certain immune deficiencies associated with the lack of antibodies.
• There are several serology techniques that can be used depending on the antibodies being studied. These include agglutination, precipitation, complement-fixation and fluorescent antibodies. • Some serological tests are not limited to blood serum, but can also be performed on other bodily fluids such as semen and saliva, which have similar properties to serum.
　

Slide Agglutination Test
• A rapid screening or semi quantitative test in which antibody and antigen are mixed on a glass slide and observed for agglutination.
• Antigen-antibody combine to form clumps which can be visualized by naked eye.
• Cannot be performed if the bacterial suspension is granular, auto agglutinates or is sticky.
• Unknown bacterial isolates are mixed with antisera to know bacteria.
• In some cases, serum from patients is mixed with known bacterial antigens to identify the antibodies in them and thus the source of infection.
• Positive slide agglutination indicates immune mediated hemolytic anemia.
• A negative result does not rule out immune disease.
　

　

Enzyme-Linked ImmunoSorbent Assay
(ELISA)

• Enzyme-Linked ImmunoSorbent Assay: is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample.
• Used as a diagnostic tool in medicine and plant pathology, as well as a quality control checks in various industries.
• It is a useful tool both for determining serum antibody concentrations and also for detecting the presence of antigen.
• ELISA can also be used in toxicology as a rapid presumptive screen for certain classes of drugs.


http://www.biotech-weblog.com/50226711/enzymelinked_immunosorbent_assay_elisa_35_years_after.php

Genotype

http://www.howstuffworks.com/dna-evidence.htm

DNA Fingerprinting
DNA Fingerprinting is a way of identifying a specific individual, rather than simply identifying a species or some particular trait. DNA fingerprinting uses a specific type of DNA sequence, known as a microsatellite, to make identification much easier.

• All of the DNA sections are contained in every cell, any piece of a person's body, from a strand of hair to a skin follicle to a drop of blood, may be used to identify them using DNA fingerprinting.
• Every organism has its individual's unique sequence of DNA base pairs determined by exposing a sample of the organism’s DNA to molecular probes.
• Large regions of our DNA that do not consist of genes and appear to serve no useful purpose.
• Some orders of DNA are repeated thus making it easier for the scientists.
• Patterns are obtained that reflects different numbers of repeats in different individuals; the length of a particular DNA fragment is a function of the number of repeats present.
• Patterns are analyzed to get a certain probability of a match.


http://departments.oxy.edu/biology/Stillman/bi221/102700/notes.htm


http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RFLPs.html

DNA sequencing of Ribosomal RNA Gene
DNA sequencing is the determination of the precise sequence of nucleotides in a sample of DNA.

• DNA sequencing has provided a new approach for studying evolutionary relationships, since :
- all organisms have a genome
- the genes that code for vital cellular functions are conserved to are markable degree through evolutionary time

• To carry out the sequencing of the organisms, scientists cut the DNA up into short fragments, sequenced these fragments simultaneously, and then assembled the entire genome by using sophisticated computer techniques to match the fragments to each other.

• DNA sequencing is also dependent on our ability to use gel electrophoresis to separate strands of DNA that differ in size by as little as one base pair.

• The gene most commonly used for this codes for the RNA in the small subunit (SSU) of the ribosome (16s rRNA)

• 16s rRNA acts like one have of two pieces of Velcro, any genes left over from matching in a given sample can be attributed to genes from previously unknown organisms.

• Some regions of this 16S rRNA are highly conserved in all organisms, whereas other regions are more variable.


Extracted from: http://www.nucleics.com/DNA_sequencing_tools/DNA_sequencing_tools.html


Extracted from: http://psychology.wikia.com/wiki/Evolution


Amino Acid Sequencing
• Comparison of amino acid sequences from proteins of different organisms is used to determine the similarity in the DNA sequences.

• Determining what conformation it adopts and whether it is complexed with any non-peptide molecules.

• The two major direct methods of protein sequencing are mass spectrometry and the Edman degradation reaction. It is also possible to generate an amino acid sequence from the DNA or mRNA sequence encoding the protein.

• Sequence information has been successfully applied to unveil structure, function, and evolutionary relationships.

• To understand the functional roles or structure families of proteins, a lot of computational methods have been developed to classify protein sequences and detect remote homology based on their sequence similarity.



　

Extracted From: http://www.mcgill.ca/sheldon/pep_seq
　
Protein Pattern Analysis
• Sequence of an uncharacterised protein translated from genomic sequences is too distantly related to any protein of known function by overall sequence alignment, but the protein function can be identified by the occurrence in its sequence of regions resembling a known functional site or a conserved protein family fragment.

• The result of analysis is set of matches with pattern or pattern portion.

• Proteins are the product of its genes and each species is expected to produce a set of unique proteins.

• Protein profiles are obtained by Polyacrylamide gel electrophoresis (PAGE) where different proteins migrate through the gel at different rate depending on their size and charge

　

Numerical Taxonomy
Numerical taxonomy is based on calculating the percentage of characteristics that two groups have in common. It is test on the characteristics, for example, the ability to form endospores, presence of flagella, the motility of the bacteria and the lactose fermentation. Numerical taxonomy is more towards the modern approaches which applies the molecular and genetic aspects of the bacteria characteristics.

Phylogenetic Hierarchy
Phylogenetics is to determine the relationships of organisms. It is done by comparing ancestors and descendants. Evolutionary relationships are represented by phylogenetic trees.

Example of phylogenetic trees:



Sources: http://commons.wikimedia.org/wiki/Image:Phylogenetic_tree.svg