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Gram Staining Technique On Unknown Bacteria Biology Essay

Morphologically talking, bacteriums come in different forms. The undermentioned bacteriums are gram positive and rod shaped: C. diptheriae, B. subtilis, and L. acidophilis. The undermentioned bacteriums are gram positive and have a cocci form: M. roseus, S. epidermidis, S. aereus, E. faecalis.The following bacteriums are gram negative and rod shaped: P. aeruginosa, B. cepacia, C. freundii, E. aerogenes, E. coli. The undermentioned bacteriums are gram negative and have a bacillus form: P. vulgaris, and S. marcescens. In order to utilize morphology as a agency to place bacteriums it important to cognize a bacterium ‘s morphological construction and this includes its form ( Keplit ) .

Biochemically talking, gram positive bacteriums possess a cytoplasmatic lipid membrane and a thick peptidoglycan bed with many cross linkages. Other common features amongst some gm positive species are the presence of capsulated polyoses and a scourge. The aforesaid peptidoglycan bed contains teichoic acids and lipids ; furthermore, their presence signifiers lipoteichoic acids. These lipoteichoic acids are, in kernel, chelating agents ( Madigan ) . Designation of gm positive bacteriums, through a biochemical position, can be achieved by detecting the presence of a thick peptidoglycan bed with cross linkages and the presence of teichoic acids.

The 16S RNA is an built-in constituent of the ribosomal RNA. Equally far as the 16S rRNA is concerned in the designation of bacteriums, the 16S rRNA can hold many sequences within a bacterial cell, and this allows for the designation of assorted types of bacterium. Recent progresss in the 16S rRNA have indicated that PCR primers have been utilized to increase the 16S rRNA cistron in order to provide information that can be used to distinguish between bacterial types. Furthermore, primers are used to analyze the 16S rRNA cistron ; besides, the 16S rRNA contains primer adhering sites. PCR is a new technique used to place bacteriums. Scientists are able to make this by placing the presence of hypervariable parts that exist within the 16S rRNA cistron sequence. These hypervariable parts contain expressed sequences that are peculiar to certain types of bacterial species. As a consequence, bacterial designation on a species broad footing can be achieved by utilizing PCR technique ( Weisburg ) .

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Respiratory beings obtain their energy through the usage of negatrons. Respiratory organisms transportation negatrons from energy beginnings to electron acceptors. These electron acceptors are so used for either aerophilic or anaerobiotic respiration ( Zillig ) . Harmonizing to Dr. Maxwell ‘s research lab notes on respiratory micro-organisms, an illustration of a respiratory micro-organism is Streptococcus pneumonia.

Materials and Methods:

Harmonizing to Dr. Maxwell ‘s research lab notes for the respiratory lab, the undermentioned stuffs were used for the respiratory lab: two unfertile swabs, one blood agar home base, one tubing of saline solution, two tubings of 9 milliliters unfertile H2O, two liquefied TSA, and two unfertile Petri dishes. The undermentioned process was completed for the respiratory lab: pupils swabbed the tonsialary part of their lab spouse by doing a peal gesture up the tonsilary part. Next, the swab was removed and inoculated onto the media. Of the two swabs that were used, the first swab was used to state if any of the civilization was haemolytic. The micro-organisms that were collected signifier the swabs were inoculated onto a blood agar home base utilizing the streaking for isolation method. This blood agar home base was so incubated in a candle jar. After the settlements were grown on the home base and stray, stray settlements were identified and checked for haemolytic activity. The stray settlements were observed and so examined for alpha haemolysis, beta haemolysis, or gamma haemolysis. Gram discolorations were so made for beta haemolytic or alpha haemolytic settlements. If a beta settlement was observed and it was gram positive, so it is likely that the being is Streptococcus pyogenes. Refering the 2nd swab, it was used for happening out the population denseness of the micro-organism from the pharynx. Aseptic techniques were used when puting the swab into a trial tubing of unfertile H2O. This trial tubing was so swirled to acquire the beings into the H2O. Next, dilutions were carried out so that there was one trial tubing that had a 10-3 dilution and the other trial tubing had a 10-4 dilution. Afterwards, a pour home base was made for both the 10-3 dilution and the10-4 dilution. After the settlements grew, they were counted to happen out the population denseness nowadays. The lab spouses so compared their several population densenesss. It is of import to observe that gm discolorations of the respiratory beings were non performed due to limited clip and the demand to originate the unknown experiment.

The followers is the dichotomous keys used to find the unknown. Note, the first trial used to place the terra incognita was the gm staining trial. Based on the consequences of the gm staining process, the bacteriums were classified as either a gram positive or gram negative bacteriums.

The undermentioned trials were completed in order to place the unknown being: Gram discoloration, lactose agitation, casein digestion, acid released signifier galactose trial, catalase trial, indole trial, glucose trial, citrate trial, growing on TSA home base trial, MR/ VP trial, and manitol trial. For the processs for the aforesaid trials are listed below ; in add-on, the beginning for these processs is from Dr. Maxwell ‘s research lab notes. The beginning for the gm staining process is Dr. Maxwell ‘s research lab notes for lab 4. The beginning for all of the staying biochemical trials is Dr. Maxwell ‘s research lab notes for lab 9.

Gram staining process:

First a bacterial vilification is created. It is of import to observe that merely a little sample of bacteriums is needed. In order to acquire a little sum of bacteriums, gently touch the inoculating cringle to the bacterial settlement. Let this vilification to air dry for about 10-15 proceedingss. After this, the vilification is heat fixed by go throughing the slide over the fire 2-3 times, and the slide is so cooled. Following, the dyes are used. The vilification is foremost covered with the primary stain- crystal violet for 60 seconds. The slide is so rinsed with H2O utilizing a rinse bottle ; in add-on the slide is rinsed until the H2O is clear or pale lavender. Afterwards, the slide is covered with the mordant, which is gm ‘s I, for 60 seconds. The slide is so rinsed with H2O. Next, the decolorizer is added and kept on the slide for less than 20 seconds, and so the decolorizer is rinsed off with H2O. Next, the counter discoloration, which is saffranine, is added. Safranin is kept on the slide for 45- 60 seconds. Safranin is so rinsed off with H2O. Afterwards, the slide is blotted with boozy paper and observed in through a microscope. Gram positive beings appear purple in colour while gram negative beings appear red in colour.

Lactose Fermentation Trial:

Using an vaccinating cringle, inoculate tubings of media with growing from an 18- 24 hr pure civilization. Incubate the tubings with disentangled caps at 35 +/- 2 grades Celsius for 48 hours in either aerophilic or anaerobiotic conditions depending on the type of bacteriums. An uninoculated tubing will be orange in colour. A typical positive reaction with acid and gas will look to be xanthous. A typical negative reaction with positive growing will look to be red/ pink.

Casein Digestion Test:

Skim milk is used to do the microbiological civilization media. The medium is heated in a boiling H2O bath for 2-5 proceedingss with the disentangled caps. It is so cooled to room temperature with the caps tightened. The tubings are so inoculated by utilizing an vaccinating cringle. As a side note, if anaerobiotic bacteriums are being investigated, unfertile mineral oil is placed over the medium after it has been inoculated. The tubings are so incubated with their caps tightly fixed for clostridium but for all other beings, the caps are loosened. The incubation temperature is 35 +/- 2 grades Celsius. Observations are made in intervals of 7 yearss. Growth and reactions are observed.

Acid released from galactose/ mannose:

Using an vaccinating cringle, inoculate tubings of media with growing from an 18- 24 hr pure civilization. Incubate the tubings with disentangled caps at 35 +/- 2 grades Celsius for 48 hours in either aerophilic or anaerobiotic conditions depending on the type of bacteriums. An uninoculated tubing will be orange in colour. A typical positive reaction with acid and gas will look to be xanthous. A typical negative reaction with positive growing will look to be red/ pink.

Catalase Test:

The bacterial population is reacted with H peroxide. An inoculating cringle is used to roll up a big sum of bacterial cells onto a microscope slide. Next the bacterial cells are smeared onto the microscopic slide. Add one or two beads of H peroxide on the vilification. If bubbles appear, the consequence is a positive reaction. If bubbles do non look the reaction is negative.

Indole Trial:

Stab the inoculating needle two-thirds of the mensural distance to the bottom centre of the tubing. Make this by utilizing growing from a pure civilization. These tubings are so incubated with their caps loosened for 18- 24 hours at 35 +/- 2 grades Celsius. It is of import to observe that aerophilic conditions are maintained when incubating.

Manitol Trial:

It is of import to obtain stray settlements. Once an stray settlement is obtained, home bases are incubated for 24- 48 hours at 35 +/- 2 grades Celsius. It is of import to observe that aerophilic conditions are maintained when incubating.

Glucose Trial:

Using an vaccinating cringle, inoculate tubings of media with growing from an 18- 24 hr pure civilization. Incubate the tubings with disentangled caps at 35 +/- 2 grades Celsius for 48 hours in either aerophilic or anaerobiotic conditions depending on the type of bacteriums. An uninoculated tubing will be orange in colour. A typical positive reaction with acid and gas will look to be xanthous. A typical negative reaction with positive growing will look to be red/ pink.

Citrate Trial:

Slants are inoculated with pure civilization. This is done by utilizing a light inoculant. Tubes are incubated for 4 yearss at 35 +/- 2 grades Celsius. It is of import to observe that aerophilic conditions are maintained when incubating.

Growth on TSA home base:

This is done simply by detecting the colour alteration and olfactory property released from the growing of bacteriums onto the TSA home base. Bacterias are grown onto a TSA home base. Once the bacterium is grown onto the home base, the colour and olfactory property are examined. A green colour and grape-like aroma indicate the presence of P. aeruginosa. If there is a clear growing with no green colour and accordingly no grape-like aroma, so this indicates the presence of B. cepacia.

MR-VP Trial:

Tubes of MR-VP media are inoculated by utilizing a light inoculant. These tubings are inoculated with 18-24 hr pure cultures ; in add-on, the tubings are held under aerophilic conditions when they are incubated at 35 +/- 2 grades Celsius for at least 48 hours. The methyl ruddy index is made by fade outing 0.1g of methyl red in 300 milliliter of 95 % ethyl intoxicant. Purified H2O is so added to do 500 milliliter. After incubation has taken topographic point, the aliquots are aseptically removed. For the methyl ruddy trial, 5 beads of methyl ruddy index are added to an aliquot of stock. The colour is so examined. For the Voges- Proskauer Test ( VP ) , 15 beads from the reagent A dropper and 5 beads from the reagent B dropper are placed into 1 milliliter of broth civilization. It is of import to agitate after each reagent is added, and this is done to air out the sample.

Consequences

For the respiratory lab

Two home bases were inoculated with bacteriums ; furthermore, these two home bases contained dilutions from the original swabs. These two dilutions included one home base that contained a 10-3 dilution and the other home base contained a 10-4 dilution. The home base with the 10-3 dilution had a “ excessively legion to number ” figure of settlement organizing units. The home base with the 10-4 dilution had 228 settlements. Each settlement, from both home bases, appeared to be really little and white.

For the unknown lab

The unknown figure was 11. When the two bacteriums were separated from each other and pure stray settlements were grown, the consequences of the gm discoloration indicated that one was gram positive ( violet in colour ) and the other was gram negative ( ruddy in colour ) . 11A will be used to depict the gm positive bacteriums, and 11B will be used to depict the gm negative bacteriums. The gm positive bacteriums, 11A, had a creamy white colour and the sizes of the bacterial settlements were average sized, but non really big. The gram negative bacteriums, 11B, had really little settlements and the colour was pink. The gm positive bacteria was cocci shaped and the gm negative bacteria was bacillus shaped.

UNKNOWN 11A

Trial

Consequence

Gram staining

Purple color= gm positive.

Lactose Agitation

Positive, xanthous colour means typical positive reaction with acid and gas.

Catalase Test

Positive, bubbles formed.

Manitol Test

Yellow zones in the center and orange zones on the sides.

Observation of colour

Very white

UNKNOWN 11B

Trial

Consequence

Lactose Agitation

Negative, ruddy colour means typical negative reaction with positive growing.

Glucose Test

Positive, xanthous colour means typical positive reaction with acid and gas.

Indole Trial

Negative, clear and no ruddy colour

UNKNOWN 3, unknown figure 113

Trial

Consequence

Gram discoloration

Gram negative rods

Lactose trial

At foremost the colour appeared to be orangish, but subsequently the colour changed to yellow, and this is a typical positive reaction with acid and gas. As a consequence of this mistake and deficiency of clip it was non possible to go on experiments to find the individuality of the unknown.

Decision:

For the respiratory being, since there was no clear zone around the bacterial settlements and non any green pigmented by-product, the respiratory being had a gamma haemolysis feature.

For the gm positive bacteria, the Lactose Fermentation trial was performed. The consequence for the gm positive being was that it appeared yellow after the Lactose Fermentation trial, and this means that it is a typical positive reaction with acid and gas. Following, the Catalase trial was performed for the gm positive being. The consequence of the Ctalase trial was that bubbles appeared and this indicates a positive consequence. Next the Manitol trial was performed on the gm positive being, and the consequence is that there were xanthous zones and an orange zone and this indicates a positive consequence for this trial. This indicated that the gm positive being is S. aereus ; in add-on, since the bacteria is really white, it can be concluded that the gm positive terra incognita is S. aerues white.

For the gm negative bacteria, the Lactose Fermentation trial was performed. The gram negative being appeared ruddy after the Lactose Fermentation trial, and this means that it is a typical negative reaction with positive growing. The Glucose trial was performed after the Lactose Fermentation trial. The consequences of the Glucose Test were that the colour observed was xanthous and this indicated a typical positive reaction with acid and gas. Next the Indole trial was performed for the gm negative bacteria. The consequences of the Indole trial show that the tubing was clear and no ruddy colour was present and this means that the consequence is negative for indole formation. Therefore, it can be concluded that the gm negative being is S. marcescens.

For the 3rd unknown, it was a gram negative being that was rod shaped. After executing the Lactose Fermentation trial, the initial consequence was that it appeared orange ( a negative consequence ) and the TA confirmed this consequence. However, one twenty-four hours subsequently the colour changed to yellow. This xanthous consequence indicates that the consequence is in fact positive with the presence of acid and gas. Due to this mistake and non adequate clip due to concluding test, the individuality of the 3rd terra incognita could non be determined. However, the undermentioned illustrates stairss that should hold been carried out. Since the Lactose Fermentation trial had a positive consequence, the following trial to be performed should hold been the Citrate trial. Once the Citrate trial is completed, a negative consequence would bespeak that the unknown bacteria is E. coli. A positive consequence for the Citrate trial would bespeak that the unknown bacteria is either C. freundii or E. aerogenes. If the consequence was positive for the Citrate trial, so the MR-VP trial would hold to be conducted in order to distinguish between C. freundii and E. aerogenes. A negative consequence for the MR-VP trial indicates that the unknown is C. freundii. A positive consequence for the MR-VP trial indicates that the unknown is E. aerogenes.

Information on S. aerues white

The followers is the taxonomy: Bacteria ; Firmicutes ; Bacilli ; Bacillales ; Staphylococcaceae ; Staphylococcus ; Staphylococcus aureus. S. aerues white is a gram positive bacteria that has a cocci form ; moreover its construction includes a thick peptidoglycan bed, cross Bridgess, and no scourge. Equally far as physiology is concerned, S. aerues white is immune to penicillin due to the production of beta-lactamase which is an enzyme that breaks down the beta-lactam ring of the penicillin molecule. S. aerues white is a facultative anaerobe which means it makes ATP by aerophilic respiration but can besides obtain energy from agitation. Its home ground includes dirt and mucose membranes of mammals. S. aerues white ‘s usage as a pathogen is merely effectual if the tegument barrier has already been broken, and one disease that it can do is known as “ boils ” ( “ Staphylococcus aureus and S. epidermis ” ) .

Information on S. marcescens

The followers is the taxonomy: Bacteria ; Proteobacteria ; Gammaproteobacteria ; Enterobacteriales ; Enterobacteriaceae ; Serratia ; Serratia marcescens. Equally far as construction is concerned, S. marcescens is bacillus molded and is a gram negative bacteria with a thin peptidoglycan bed. Equally far as physiology is concerned, S. marcescens can release the heme-binding protein HasA. S. marcescens is a facultative anaerobe which means it makes ATP by aerophilic respiration but can besides obtain energy from agitation. Its home ground includes life in the dirt, H2O, and bowels of mammals. Its usage as a pathogen includes its function in nocosomial infection ; moreover it is known to do urinary and respiratory piece of land infections ( “ Serratia marcescens ” ) .

Beginnings:

Keplit, E. ( n.d. ) . Bacterias: more on morphology. Retrieved from hypertext transfer protocol: //www.ucmp.berkeley.edu/bacteria/bacteriamm.html

Madigan M ; Martinko J ( editors ) . ( 2005 ) . Brock Biology of Microorganisms ( 11th ed. ) . Prentice Hall.

Serratia marcescens. ( n.d. ) . Retrieved from hypertext transfer protocol: //www.sunysccc.edu/academic/mst/microbes/23smarc.htm

Staphylococcus aureus and S. cuticle. ( n.d. ) . Retrieved from hypertext transfer protocol: //www.sunysccc.edu/academic/mst/microbes/13saure.htm

Weisburg, WG. , Barns, SM. , Pelletier, DA. , & A ; Lane, DJ. ( 1991 ) . 16s ribosomal deoxyribonucleic acid elaboration for phyletic survey. Journal of Bacteriology, 2, 697-703.

Zillig W ( 1991 ) . “ Comparative biochemistry of Archaea and Bacteria ” . Curr Opin Genet Dev 1 ( 4 ) : 544-51.

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