Identification of Gram Negative Bacteria (Lab Report Sample)

Identification of Gram Negative Bacteria
Laboratory Report
Name
Module
Date
Abstract
The main objective of this experiment was to distinguish between Escherichia coli and Klebsiella pneumoniae cultures. The system of distinguishing the two involved using morphological characteristics and biochemical tests. Morphological characteristics that were exhibited in this experiment were colony characteristics and motility. E. coli colonies were found to have rough margins while those of K. pneumonia had smooth edges. E. coli lacked the capability to utilize citrate under anaerobic conditions. The ability of bacteria to develop resistance to antibiotics was highlighted. The main reason for the emerging mutations and consequent resistance to drugs is the overuse of antibiotics. It was agreed that the best method to tell the two species apart was by using quick methods such as motility. Other methods such as citrate utilization are expensive and not capable of delivering producible results.
Identification of Gram Negative Bacteria.
Laboratory Report.
Background
The main difference between gram- positive and gram- negative bacteria is there appearance below the light microscope after performing the gram staining procedure. At X100 magnification with the aid of immersion oil, gram -positive bacteria appear to dark blue due to the crystal violet stain. Gram- negative bacteria on will appear red because they take up the safranin counter stain. The primary principle behind these differences is due to the difference in the thickness of the peptidoglycan layer of these bacteria (Claus, 1992). Gram- positive bacteria have a thicker peptidoglycan layer and are therefore able to retain the crystal violet stain once washed with ethyl alcohol. Gram- negative bacteria have a thinner peptidoglycan layer that cannot withstand a wash by ethyl alcohol. When safranin is applied to both groups, the Gram- positive bacteria will not take it up because its peptidoglycan is already saturated with crystal violet. On the other hand, the gram- negative bacteria will readily take up the counter stain because the primary stain (crystal Violet) was washed off with ethyl alcohol.
Due to the importance associated with their identification, it is crucial that students acquire skills necessary for telling apart these bacterial groupings. Students ought to learn how to use various biochemical tests to identify gram- negative bacteria to the species level. More often than not, it requires a lot of practice to acquire such skills. A misidentification of a gram- negative species can turn fatal or ineffective in the event that the doctor prescribes specific antibiotics following a false laboratory finding. Experiments such as this one are fundamental in imparting basic laboratory skills. Students also gain first -hand experience unlike when they read these tests in books. In this regard, it is important that students learn about the biochemical tests that are currently in use during the identification of gram- negative bacteria species. This particular experiment encompasses a set of biochemical tests that distinguish between various gram- negative bacteria such as Glucose Oxidation Function, indole utilization, and citrate utilization. Other features used to tell apart gram -negative bacteria include morphology, anaerobic growth and colony characteristics.
Materials and Methods
Materials
1 Cultures- Escherichia coli and Klebsiella pneumonia, Moraxella as a control and young peptone water cultures for motility tests.
2 Oxidase strips
3 Sterile toothpicks
4 Reagents for MR and VP tests
5 Peptone water
6 Urea agar slopes
7 Kligler iron agar
8 Simmons citrate medium and sterile saline
9 Ornithine and lysine decarboxylase medium and base broth + sterile paraffin
10 Indole reagent and filter paper strips.
Identification tables for bacteria.
TEST

E.coli

K. pneumoniae

Colony features
NA/HBA
MCA
Anaerobic growth

O2, 370C, 24 hours (Conditions)
Large colonies
Irregular margins
Opaque
Flat
Glossy/ shiny
O2, 37OC, 24 hours (conditions)
Large
Pink
Flat
Yes

O2, 37oC, 24 hours
Large colonies
Smooth margins
Transparent
Elevated
O2, 37OC, 24 hours
Comparatively larger
pink
Lighter center
Not tested.

Gram morphology

Gram- negative rods

Gram- negative rods

Glucose OF
Acidic aerobically
acidic anaerobically
energy metabolism

Positive
Positive
anaerobic


oxidase

No color change

No color change

Motility

yes

no

Indole production
Conventional test
Rapid spot test

Positive

Negative

Citrate utilization

negative

positive

Kligler
Acid from glucose
Acid from lactose
Gas production
H2s production

Positive
Positive
Positive
Negative

Positive
Positive
Positive
negative

Urease

negative

positive

Methy red
(pH<2)

Positive

Negative

Voges- proskauer
(acetoin production)

positive

Negative

Lysine decarboxylase

positive

Negative

Ornithine decarboxylase

Positive

Negative

The results above show the numerous tests that are used to distinguish between E. coli and K. pneoumoniae. One cannot distinguish between the two using a simple gram staining procedure because they are all gram- negative. When growing on NA or HBA, an interested party can distinguish the two in that E. coli has flat colonies while those of K. pneumonia are elevated. When growing on MCA, it will be impossible to distinguish between the two according to the findings of this particular study. When growing in young peptone water, E.coli cells are capable of motility while those of K. pneumoniae are non- motile.
Biochemical tests in which E.coli tests positive while K. pneumoniae test negative are as follows. Ornithine decarboxylase, lysine decarboxylase, Voges- proskauer (acetoin production), Methyl red (pH<2) and indole production. The following list comprises of the biochemical tests in which K. pneumoniae tested positive and E.coli tested negative. Urease and Citrate utilization.
Discussion
When E.coli cells are put in young peptone water, they appear to be motile when an observation is made under high power magnification. E. coli cells are able to move because they can develop filaments on their sides (Maki et. al, 2000). Comparatively, K. pneumoniae does not have the capability of developing filaments hence the lack of motility. Filamentous cells of E.coli are produced when the bacterium is in an environment that inhibits growth such as in the presence of antibiotics. The filamentous cells are chemotactically attracted or brepelled by different substances. When in the presence of a chemo- attractant, filaments respond by prolonged running towards it. When faced with a chemo- repellant, the filaments exercise prolonged stopping to reduce any chances of injury to the cell. Apart from the filamentous cells, all E.coli cells have six flagella to propel them in any direction. When the flagella rotate anticlockwise, the form a bundle on one end and push the cell forward (run). When rotating in the clockwise direction, flagella on each end pull in the opposite direction making the cell to tumble.
Unlike K. pneumonia, E. coli is usually unable to utilize citrate in the presence of oxygen (Evo.ed, 2015). However, under aerobic conditio...