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Microbiology-microorganism identification - Assignment Example

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Most “cocci” which appear Gram negative are in fact short rods, whilst “true” cocci in this exercise are all Gram positive. When you have completed the table of…
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Microbiology-microorganism identification
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Results – Lab #6: The identification of bacteria Bergey’s Manual DIAGNOSTIC TABLE: Please that many characters can vary with strain type. Characters* = Most Strains
Litmus Milk: Acid (A); Clot (C)
All the unknowns used in this laboratory are presented.
Organism
Pigment Production
Cell Shape
Cell Arrangement
Gram Stain
Spore Production
Motility
Opt. Growth Temp.oC
Aerobic Growth
Anaerobic Growth
Catalase
Oxidase
Oxidative
Fermentative
Citrate utilization
Nitrate production
Indole Production
Glucose fermentation
Lactose fermentation
Sucrose fermentation
Methyl Red (MR)
Voges-Proskauer (VP)
Gelatin Hydrolysis
Litmus Milk
Bacillus subtilis
_
R
Seldom in chains
+
+
+
37
+
_
+
*_
+
+
_
+
_
_
_
_
+
*C
Enterobacter aerogenes
_
R
Single
_
_
+
37
+
+
+
_
+
+
+
_
+
+
+
_
+
*+
A
Escherichia coli
_
R
Single
_
_
+
37
+
+
+
_
+
_
+
+
+
+
+
*+
_
_
Klebsiella pneumoniae
_
R
Single or pairs
_
_
_
37
+
+
+
_
+
*+
+
_
+
+
+
*_
+
_
Micrococcus luteus
Y
C
Clusters
+
_
_
30
+
_
+
_
+
_
_
_
_
_
_
_
_
*+
Proteus vulgaris
_
R
Pairs or chains
_
_
+
* 37
+
+
+
_
+
+
+
_
_
+
+
_
+
Pseudomonas aeruginosa
G
R
Single
_
_
+
37
+
_
+
+
+
+
+
_
_
_
_
_
_
+
Serratia marcesens
R
R
Single
_
_
+
30
+
+
+
_
+
+
+
_
+
_
+
*+
*+
+
*A
Staphylococcus aureus
*_
C
Clusters
+
_
_
37
+
+
+
_
+
+
_
+
+
*+
+
+
+
A
Enterococcus faecalis
_
C
Single, pairs, short chains
+
_
_
37
+
+
_
_
+
*+
_
_
+
+
+
+
_
_
A
KEY:
Pigment Colour: Yellow (Y); Red (R); Green (G)
Cell Shape: Rod (R); Cocci (C)
Observed characteristics
NOTE: Many Gram negative rods can be very short and appear almost coccoidal under some growth conditions. Most “cocci” which appear Gram negative are in fact short rods, whilst “true” cocci in this exercise are all Gram positive. When you have completed the table of characters and are confident that you have the correct Gram stain result and shape THEN refer to the key (p33 & p34 of the Procedures Manual) and diagnostic table (p35 of this Results Manual) to Identify your Unknown.
CULTURE #
COLONIAL MORPHOLOGY ON NUTRIENT AGAR
Pigment Production
CELL MORPHOLOGY
Shape of Cells
Arrangement of Cells
Gram Stain
Spore Production
Motility
PHYSIOLOGICAL & BIOCHEMICAL CHARACTERISTICS
Optimum Growth temperature
Oxygen requirements
Catalase
Oxidase
Oxidation/Fermentation
Citrate Utilization
Nitrate Reduction
Indole Production
Fermentation of Glucose
Lactose
Sucrose
Methyl Red
Voges-Proskauer
Litmus Milk
Gelatin Hydrolysis
IDENTITY
CONCLUSIONS & DISCUSSION
After identifying your unknown as closely as possible and with reference to Bergy’s Manual answer the following questions.
1. Which characteristics were the most significant for the correct identification of your organism?
The most significant characteristics for the correct identification of our organism included the ability to stain positive using the Gram stain. From the diagnostic table, only four bacterial species are expected to test positive using Gram stain, namely Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus and Enterococcus faecalis. The Gram stain test thus allows the analyst to reduce the number of possible bacterial species that need to be ruled out during the identification assay. Another characteristic that facilitates our species identification was associated with the positive results obtained from the methyl red (MR) test. The observation of a bright red color was instrumental in narrowing down our candidates species to two bacteria, namely Staphylococcus aureus or Enterococcus faecalis. The generation of negative results from the Gelatin Hydrolysis, Voges-Proskauer (VP) and Nitrate Production tests also assisted in the identification of bacterial species.
2. Did all the observed characteristics agree with those in Bergey’s Manual? Note and explain any of which did not.
No, not all the observed characteristics agreed with those indicated in Bergey’s Manual. The cell arrangement of our organism was observed to be in single pairs or short chains, while the manual described that the bacteria were generally found in clusters. In addition, a positive result was observed in the citrate utilization test, when the manual indicated that our bacterial species generally results in a positive reaction.
Unfortunately, the catalase and oxidase tests were not performed due to lack of time. The microbiological assay involves the reaction of the catalase enzyme, as it disrupts hydrogen peroxide, resulting in the production of water, as well as oxygen. Catalase is considered as a very common protein enzyme that is present in almost of organisms and is regarded by microbiologists as a very useful enzyme that helps in the identification of specific bacterial species (Maehly and Chance, 1954). It is also possible to identify the bacterial species through its requirement for oxygen. It should be understood that both oxidation and fermentation tests generated positive results and the next essential procedure that can be performed in the determination for the presence of catalase.

3. Recommend any further NON-culture based tests that could be carried out to confirm the identity of your unknown.
Another non-culture based test that could be carried out to confirm the identity of our unknown is the polymerase chain reaction (PCR). In the PCR assay, the DNA of the bacterial cells are extracted and subjected to an enzymatic reaction that would generate significant amounts of DNA of a specific DNA region of interest. The PCR test can be designed to target specific-specific sequences that are uniquely present in this bacterial strain. This assay does not involve culturing of the cells and would only require direct DNA extraction from the isolates.
4. FULLY discuss the importance of the accuracy of bacterial identification:
Bacterial identification is important in every microbiological laboratory because it provides the analyst information on how to handle the microorganism. The proper identification is also essential most microbiological laboratories because it facilitates in the design of the treatment regimen that should be performed on a patient who is suffering from a bacterial infection. In order to achieve accurate bacterial identification of isolates, a number of procedures should be implemented in the laboratory. One of the most important principles is that the laboratory should be maintained in the most aseptic condition and that any contamination with other isolates and existing laboratory personnel should be avoided. The microbiology laboratory should also follow a systematic method in labeling and tracking down the case sample within the laboratory. There are currently a number of microbiological tests that have been considered as gold standards in the identification of bacterial isolates. Aside from the simple microscopic descriptions of the basic cellular morphology of the bacteria, such as rod- or spherical-shaped, there are also staining assays that could facilitate in the identification of the bacterial isolate. The Gram staining test allows the classification of a specific group of bacteria, namely the Gram-positive and the Gram-negative strains. Additional biochemical tests can then be performed on the Gram-stain classified bacteria, based on their reactions to specific enzymes such as catalase, oxidase and other enzymes.
The accurate identification of a bacterial species is important because it allows the analyst information on how to treat a patient with the proper antibacterial drug. The correct identification of the bacteria also provides information of how to prevent its further spread across the city, thus preventing an outbreak or an epidemic. The proper identification of a bacterial species will also provide information of whether a species is resistant to specific antibiotics. It should be understood that multi-drug resistance is currently a global health issue due to the improper administration and use of antibiotics for several decades. The determination of multi-drug resistance in a specific bacterial strain will provide information to the physician on which antibiotic should be administered to a patient.
Reference
Maehly, A. and Chance, B. (1954). The assay of catalases and peroxidases. Methods in Biochemical Analysis, 1, 357–424. Read More
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