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Tests to Identify Bacteria

Introduction The purpose of this lab was to identify unknown bacteria cultures using various differential tests. The identification of these unknown cultures was accomplished by separating and differentiating possible bacteria based on specific biochemical characteristics. Whether the tests performed identified specific enzymatic reactions or metabolic pathways, each was used in a way to help recognize those specifics and identify the unknown cultures. To initiate differential testing of unknown microorganisms, the Gram stain procedure was performed. The art of Gram staining is important because it allows students, as well as microbiologists, to differentiate between bacteria and the human cell. Along with revealing the differences between bacteria and the human cell, Gram stain, also divided bacterial cells into two different groups known as Gram positive and Gram negative. While revealing the identity of cells, Gram staining also explains differences between different types of bacteria and shapes specific to them. Bacteria are usually composed of either cocci, or sphere shaped, bacillus, or rod shaped, and spirillum, which are helical and curved.
More recent advances have aided in identifying the morphology and identification of microorganism. One known method is the 16S rRNA gene sequencing. 16S rRNA gene sequences contain hypervariable regions which can provide species-specific signature sequences useful for bacterial identification. As a result, 16S rRNA gene sequencing has become prevalent in medical microbiology as a rapid, accurate alternative to phenotypic methods of bacterial identification (Smit and etl 2007).
In this unit, students also focused on the pathogens of the respiratory tract and harvested the flora of the throat by inoculating the sample onto a blood agar media. Viruses are distinctive agents that cause inflammation of the throat (pharynx) and because of this there are a number of bacteria that can cause pharyngitis. The three most common causes of pharyngitis are Streptococcus pneumonia, Haemophilus influenza, and Moraxella catarrhalis. Less common, but more severe is streptococcal pharyngitis, or strep throat, caused by Streptococcus pyogenes. The pharynx has a normal flora, a community of organisms that commonly inhabit the pharyngeal tissues. This experiment was designed to show the presence of hemolytic organisms and their ability to destroy red blood cells. Even though hemolysis is a common characteristic among upper respiratory pathogens, there are different types of hemolysis (Maxell 2008).
The differential tests used to identify the first unknown culture were Catalase test and Mannitol Salt Agar for the gram-positive and the Simmons Citrate Slants, Glucose Fermentation and the Lactose test for the gram-negative organisms. The unknown #102 culture was revealed by using the Catalase test, Mannitol Salt agar, and Lactose test.
Material

Identification of Micro-Organisms Assesment

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

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