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Identification of Micro-Organisms Assesment

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With Bacteria as can be seen by the following table a process of elimination can be utilised to identify a previously unknown micro-organism but it is a time consuming and potentially costly process.

In the Pathology Laboratory set up clinical information from the clinical staff would give the technical staff pointers to narrow the field and facilitate a prompt identification so that early treatment can be instigated.

Gram staining.

+

+

+

+

+

 

+

+

+

+

Morphology.

cocci (clusters)

cocci (clusters)

cocci (chains)

cocci (tetrads)

rod

rod

irreg. rods

rod

rod

rod

rod

rod

rod

rod

rod

cocci (pairs)

Aerobic growth

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

Anaerobic growth

+

+

+

+

+

+

+

+

+

Endospores

+

+

+

Motility.

+

+ or–

+ or–

+ or–

+ or–

+ or–

+

+

Catalase reaction

+

+

+

+

+

+

+

+

+

+

+

+

Benzidine reaction

+

+

+

+

+

+

+

+

+

+

+

+

Oxidase reaction

+

+ or–

+ or–

+

+

+

+

Glucose fermentation to acidorto acid+gas

+

+

+

+

+(or –)

+

+

+

+

Micrococcus

X

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Staphylococcus

 

X

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Streptococcus

 

 

X

 

 

 

 

 

 

 

 

 

 

 

 

 

Lactococcus

 

 

X

 

 

 

 

 

 

 

 

 

 

 

 

 

Enterococcus

 

 

X

 

 

 

 

 

 

 

 

 

 

 

 

 

Leuconostoc

 

 

X

 

 

 

 

 

 

 

 

 

 

 

 

 

Pediococcus

 

 

X

X

 

 

 

 

 

 

 

 

 

 

 

 

Aerococcus

 

 

 

X

 

 

 

 

 

 

 

 

 

 

 

 

Lactobacillus

 

 

 

 

X

 

 

 

 

 

 

 

 

 

 

 

Kurthia

 

 

 

 

 

X

 

 

 

 

 

 

 

 

 

 

Arthrobacter

 

 

 

 

 

 

X

 

 

 

 

 

 

 

 

 

Clostridium

 

 

 

 

 

 

 

X

 

 

 

 

 

 

 

 

Bacillus

 

 

 

 

 

 

 

 

X

X

 

 

 

 

 

 

Alcaligenes

 

 

 

 

 

 

 

 

 

 

X

 

 

 

 

 

Pseudomonas

 

 

 

 

 

 

 

 

 

 

 

X

 

 

 

 

Klebsiella

 

 

 

 

 

 

 

 

 

 

 

 

X

 

 

 

Shigella

 

 

 

 

 

 

 

 

 

 

 

 

X

 

 

 

Salmonella

 

 

 

 

 

 

 

 

 

 

 

 

 

X

 

 

Escherichia

 

 

 

 

 

 

 

 

 

 

 

 

 

X

 

 

most other enteric genera

 

 

 

 

 

 

 

 

 

 

 

 

 

X

 

 

Aeromonas

 

 

 

 

 

 

 

 

 

 

 

 

 

 

X

 

Chromobacterium

 

 

 

 

 

 

 

 

 

 

 

 

 

 

X

 

Neisseria

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

X

Microscopic identification of medically significant fungi follows:

Key features for identification include:

  • Coenocytic, mostly non-septate hyphae.
  • Zygospore morphology in homothallic strains.
  • However most isolates are heterothallic, i.e. zygospores are absent, and therefore identification is based primarily on sporangial morphology.

Key Features include Microscopic Morphology and Culture Characteristics:

It is mandatory to see conidial characteristics to make a precise identification therefore you must have a good slide. A conidia is a spore produced asexually by various fungi at the tip of a specialized hypha.

Preparation [needle mounts, tape mounts, slide cultures]. It may also be required to stimulate sporulation by using different media. If conidia are present then assess the following characters:

1. Conidial characteristics:

Septation.

Shape [spherical, subspherical, pyriform, clavate, ellipsoidal etc].

Size [need graduated eye piece, <10 mm etc].

Colour [hyaline or darkly pigmented].

Wall texture [smooth, rough, verrucose, echinulate etc].

How many conidial types present? [Micro and macro].

(2) Culture Characteristics.

These can also assist in fungal identification but are less reliable as the media and growth conditions play an important part.

Examine the following characteristics:

Surface texture [glabrous, suede-like, powdery, granular, fluffy, downy, cottony etc].

Surface topography [flat, raised, heaped, folded, domed, radial grooved].

Surface pigmentation [white, cream, yellow, brown, green, grey, black etc].

Reverse pigmentation [none, yellow, brown, red etc].

Growth rate [eg colonies growing less than 5 mm in 14 days etc].

Growth temperature studies are also often very useful [37oC, 40oC & 45oC].

An accurate combination of Cultural & Conidial characteristics make fungal identification a precise science.

REF: Brock: Biology of Micro-Organisms 8th Edition Madigan Martinko and Parker.

Southern Illinois University

Identification of Viruses:

Given their small size electron microscopy is required to make any identification based purely on morphology.

  1. Virus morphology
    1. Viruses, as viewed through the electron microscope , come in a variety of shapes that may be divided into:
      1. helical viruses
      2. polyhedral viruses
      3. enveloped viruses
      4. enveloped polyhedral viruses
      5. complex viruses
  2. Helical viruses
    1. Helical virusesare nonenveloped with capsomeres which are arranged helically around the virus genome.
    2. That is, like a spiral staircase or, better, one of those helical parking lot ramps, with the genome arranged up the hole in the middle.
    3. See Tobacco Mosaic Virus.
  3. Polyhedral viruses [icosahedral]
    1. Polyhedral virusesare nonenveloped viruses whose capsids form geometric shapes with flat sides (i.e., faces) and edges.
    2. Icosahedral:
      1. An example is anicosahedronwhich has 20 equilateral triangle faces and 12 corners.
      2. Though apparently complex, a very large number of otherwise unrelated viruses areicosohedral.
      3. See Reovirus, Adenovirus, and Picornavirus.
  4. Enveloped helical virus
    1. Enveloped helical virusesare enveloped viruses whose envelope surrounds a capsid withhelical virusmorphology.
    2. See Paramyxovirus.
  5. Enveloped polyhedral virus
    1. Enveloped polyhedral virusesare enveloped viruses whose envelope surrounds a capsid with polyhedral virus morphology. E.g. Herpesvirus and Togavirus.
  6. Complex virus
    1. The morphology ofcomplex virusesconsists of complex combinations of structures that may or may not be completely consistent between viruses of the same species.
    2. Tailedbacteriophagearecomplex viruses.

Reference for Viral types: Stephen T. Abedon ([email protected])

M4 Outline how the techniques to identify Micro-Organisms relate to their structure:

Diagram Courtesy Brock’s Micro-Biology edition 8.

Using the standard light and binocular microscopes some degree of morphological taxonomy of micro-organisms is possible e.g. Cocci (D) Assorted Rods (A) Spirochetes (E) Vibrio’s (F) as in the illustration above coloured to facilitate differentiation. But this has limited value.

Our entire perception of gram-positive and gram-negative walls ultimately relies on the response of bacteria to Gram staining. Christian Gram developed a staining regimen for light microscopy which differentiated between these two types of bacteria because of the chemical composition and structural format of their cell walls. Because gram-negative bacteria possess a lipid-rich outer membrane (as well as a plasma membrane) and a thin peptidoglycan layer, the alcohol decolorizing step of Gram staining washes the primary stain (crystal violet) from the cells and the secondary stain (carbol fuchsin or saffranin) colours the bacteria red. Gram-positive bacteria are enshrouded in thicker, more resilient cell walls which do not allow the crystal violet to be removed and, accordingly, remain purple. Although the vast majority of bacteria adhere to the colour differentiation of the Gram stain, some bacteria do not. These are called gram-variable bacteria.

The stereoscopic or binocular microscopeis anopticalmicroscopevariant designed for low magnification observation of a sampleusingincident light illumination rather thanTrans -illumination. Ituses two separate optical paths with two objectives and two eyepieces to provide slightly different viewing angles to the left and right eyes. In this way it produces athree-dimensionalvisualization of the sample being examined. Stereomicroscopy overlapsmacrophotographyfor recording and examining solid samples with complex surfacetopography, where a three-dimensional view is essential or where comparative study is desired, in forensic cases e.g. Bacterial comparison.

Generally speaking, it is theoretically and practically possible to see living and unstained bacteria with compound light microscopes, including those microscopes which are used for educational purposes in schools. There are several issues to consider, however.

Bacteria are difficult to see with a bright-field compound microscope for several reasons:

  • They are small: In order to see their shape, it is necessary to use a magnification of about 400x to 1000x. The optics must be good in order to resolve them properly at this magnification.
  • Difficult to focus: At a high magnification, the bacterial cells will float in and out of focus, especially if the layer of water between the cover glass and the slide is too thick.
  • They are transparent: Bacteria will show their colour only if they are present in a colony. Individual cells present on the slide are clear. Regular bright-field optics will only show the bacteria if one closes the condenser iris diaphragm. This is due to the difference in the refractive index between the water and the bacterial cells.
  • Difficult to recognize: An untrained eye may have problems differentiating bacteria from small dust and dirt which is present on the slide. Some bacteria also form clumps and therefore it is difficult to see the individual cells.

Research organizations and advances amateurs use phase contrast optics to see bacteria. This system converts the differences of the refractive index of the bacteria into brightness. The transparent bacteria can then be seen dark on bright background. In bright-field, closing the condenser iris diaphragm will also make the bacteria appear darker, but at the same time one also introduces artefacts around the individual cells. One possibility is to stain the bacteria, but in this case there fixing and staining process may introduce artefacts.

Electron microscopy (EM) is at the highest-resolution limit of a spectrum of complementary morphological techniques. When combined with molecular detection methods, EM is the only technique with sufficient resolution to localize proteins to small membrane subdomains in the context of the cell. Recent procedural and technical developments have increasingly improved the power of EM as a cell-biological tool. Whilst this tool has some place in identification of smaller bacteria it is more useful in the identification of viruses, prions and similar micro-organisms.

EMs are capable of imaging at a significantly higherresolutionthanlight microscopes, owing to the small wavelengthof electrons. This enables the instrument's user to examine fine detail—even as small as a single column of atoms, which is thousands of times smaller than the smallest resolvable object in a light microscope. EM forms a major analysis method in a range of scientific fields, in both physical and biological sciences. EMs find application incancer research,virology,materials scienceetc.

The membrane filtration test is a fast, simple way to estimate bacterial population in water. In the initial step, an appropriate sample volume is passed through a membrane filter with a pore size small enough (0.45µm) to retain the bacteria present. The filter is then placed on an absorbent pad (in a petri dish) saturated with a culture medium that is selective for coliform growth. The petri dish is inverted and placed in an incubator for 24hrs at the appropriate temperature. After incubation, the colonies that have grown are identified and counted with a magnifying glass or light microscope at low power magnification.

Reference(s):

http://www.pall.com/main/laboratory/literature-library-details.page?id=7290

http://web.mit.edu/watsan/methods_microbiological.html

Streak platingis a technique used to isolate a purestrainfrom a single species of bacteria. Samples can then be taken from the resulting colonies and a microbiological culturecan be grown on a new plate so that the organism can be identified, studied, or tested.

The modern streak plate method has progressed from the efforts byRobert Kochand other microbiologists to obtainmicrobiological culturesof bacteria in order to study them. The dilution or isolation by streaking method was first developed by Loeffler and Gaffky in Koch's laboratory, which involves the dilution of bacteria by systematically streaking them over the exterior of the agar in apetri dishto obtain isolated colonies which will then grow into quantity of cells, or isolated colonies. If the agar surface grows microorganisms which are all genetically the same, the culture is then considered as a microbiological culture.

This technique bears little relation to the cellular structure of the micro-organism concerned however.

D4 Outline the potential usefulness of a variety of identification techniques in a specific application:

Returning to the identification table described earlier (and reproduced again below,) we can see that a number of bacteria responsible for disease in human beings that require specific identification to facilitate diagnosis and appropriate therapy.

Gram staining.

+

+

+

+

+

 

+

+

+

+

Morphology.

cocci (clusters)

cocci (clusters)

cocci (chains)

cocci (tetrads)

rod

rod

irreg. rods

rod

rod

rod

rod

rod

Aerobic growth

+

+

+

+

+

+

+

+

+

+

+

Anaerobic growth

+

+

+

+

+

+

Endospores

+

+

+

Motility.

+

+ or–

+ or–

+ or–

+ or–

+ or–

Catalase reaction

+

+

+

+

+

+

+

+

Benzidine reaction

+

+

+

+

+

+

+

+

Oxidase reaction

+

+ or–

+ or–

+

+

Glucose fermentation to acidorto acid+gas

+

+

+

+

+(or –)

+

Micrococcus

X

                     

Staphylococcus

 

X

                   

Streptococcus

   

X

                 

Lactococcus

   

X

                 

Enterococcus

   

X

                 

Leuconostoc

   

X

                 

Pediococcus

   

X

X

               

Aerococcus

     

X

               

Lactobacillus

       

X

             

Kurthia

         

X

           

Arthrobacter

           

X

         

Clostridium

             

X

       

Bacillus

               

X

X

   

Alcaligenes

                   

X

 

Pseudomonas

                     

X

Klebsiella

                       

Shigella

                       

Salmonella

                       

Escherichia

                       

most other enteric genera

                       

Aeromonas

                       

Chromobacterium

                       

Neisseria

                       

Consider Clostridium for example. This organism if it enters a wound can cause Gangrene a condition that occurs when body tissue dies. It is caused by a loss ofbloodsupply due to an underlying illness, injury, and/or infection. Fingers, toes, and limbs are most often affected, but gangrene can also occur inside the body, damaging organs and muscles. There are different types of gangrene and all require immediate medical attention.

A swab sample presented to the laboratory would be accompanied by a form containing in addition to the patients details a brief clinical description of what the sample might be e.g. pus from a wound in a patient with Diabetes Mellitus and a brief clinical report.

This will give the Technician a head start on which tests to run to identify the species involved.

Reference: Google Search.

Images of Clostridium botulinum.

This Gram positive rod is an anaerobe has an ambivalent result on motility testing and is Catalase Benzidine and Oxidase negative and when fermented with Glucose it produces acid but no gas and as can be seen in the illustration above the bacteria contains endospores.

Having identified the micro-organism treatment can follow

One type of gangrene is seen in appendicitis and whilst the initial diagnosis is usually based on clinical findings and haematological investigations the excised organ will be screened in the laboratory and culture and sensitivity tests made available in case of post-operative infection.

Gram staining is already described in the above example.

Streak plating would facilitate sensitivity testing of an organism as samples of antibiotics can be placed onto an agar plate to recommend an appropriate anti-microbial therapy .See the example below where two Streptococcal species possibly from a throat swab were cultured and tested for Bacitracin sensitivity. S. pyogenes was affected by the antibiotic whereas S. agalactiae was resistant so an alternative anti-microbial therapy would be required. Multiple discs for several antibiotics can be tested on the same agar plate thus giving a rapid result to allow the medical team to commence treatment.

Continued on succeeding page:

Reference: Photograph ASM Microbel.library.org ©Chamberlain.

Membrane filtration method:

  • Permits testing of large sample volumes.
  • Reduces preparation time as compared to many traditional methods.
  • Allows isolation and enumeration of discrete colonies of bacteria.
  • Provides presence or absence information within 24 hours.
  • Effective and acceptable technique. Used to monitor drinking water in government laboratories.
  • Useful for bacterial monitoring in the pharmaceutical, cosmetics, electronics, and food and beverage industries.
  • Allows for removal of bacteriostatic or bactericidal agents that would not be removed in Pour Plate, or Spread Plate techniques so eliminating false negative results.

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