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How to Write a Microbiology Unknown Paper | Lab Report

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 UNKNOWN LAB REPORT

Unknown Number 118 – Gram Positive

Alternate Number 1 – Gram Negative

Susan Berberich

Biology 203 – Microbiology

Fall/2013

 Introduction:

It has been estimated that there are 5 nonillion or 5 x 1030 bacteria on earth today. (1)  Identification is key to know exactly how a particular bacteria should be treated or if treatment is necessary at all.  The following study was performed using all of the methods used in general microbiology laboratory class.




 

Materials and Methods

Day 1 – October 22, 2013

Unknown mixed culture bacteria sample number 118 was received from the lab instructor.  The Lab Manual for General Microbiology – Biology 203 by McDonald et. al (2) was followed for all procedures performed.  The first procedure performed was an isolation streak as described in the lab manual on a nutrient agar plate.  Three isolation streaks were performed from mixed culture.  These were identified as Berb#1, Berb#2 and Berb#3.  The agar dishes were put in the incubator and left for 48 hours.

Day 2 – October 24, 2013

Agar dishes were removed from the incubator and examined.  The following results were noted:

Berb#1 – Isolated colonies – growth of one color

Berb#2 – Isolated colonies – growth of one color

Berb#3 was determined to be the best option distinctly different colonies.  These were named Light#1 and Creamy#2.  Light#1 – light almost transparent growth.  Creamy#2 – darker off white, creamy appearance growth. Two new agar plates (Light#1 and Creamy#2) were inoculated and placed in the incubator for 120 hours.

Day 3 – October 29, 2013

Reviewed growth on agar plates Light#1 and Creamy#2.  Visually both plates had growth that appeared different per plate.  The next step was to perform a Gram Stain of the two different samples following the procedures listed in the lab manual.  The purpose of the Gram Stain was to see if the samples were Gram Positive (purple) or Gram Negative (red).  Bacteria shape was also noted during the process.  The following results were noted:

Light#1 – Gram Positive – Coccus

Creamy#2 – Gram Positive – Coccus and Gram Negative – Bacillus

A second Gram Stain of Creamy#2 was performed with the following results:

Creamy#2 – Gram Positive – Coccus and Gram Negative – Bacillus

With mixed results from sample Creamy#2, a new agar plate was inoculated from streak plate Berb#3.  Original agar plate Creamy#2 was thrown away and a new Creamy#2 was placed in the incubator for 48 hours.

Day 4 – October 31, 2013

Reviewed growth on agar plate Creamy#2.  Performed a Gram Stain with the following results:

Creamy#2 – Gram Negative – Rods

Threw all agar plates away except :

Berb#3 – Original streak plate for colony isolation.

Light#1 – Gram Positive Cocci Bacteria

Creamy#2 – Gram Negative Rod Bacteria

Reviewed the Unknown Chart provided by the lab instructor which listed possible organisms and corresponding test results.  Based on this chart, the following agar plate tests were ran to narrow the possibilities of which Gram Positive and which Gram Negative made up the mixed unknown culture.

Mannitol Salt Agar (MSA) – Mannitol fermentation – check for acid production.  Plates labeled MSA Light#1 and MSA Creamy#2

Eosin Methylene Blue (EMB) – Lactose fermentation – check for acid production.  Plates labeled EMB Light#1 and EMB Creamy#2

MacConkey Agar – Lactose fermentation – check for acid production.  Plates labeled Mac Light#1 and Mac Creamy#2

Sample plates Berb#3, Light#1 and Creamy#2 were placed in the refrigerator.  Test plates were inoculated according to lab manual instructions and placed in the incubator for 120 hours.

Day 5 – November 5, 2013

Reviewed Gram Positive agar plates.  Based on the Unknown Chart provided there were three Gram Positive Cocci possibilities:  Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis. The test results were:

MSA Light#1 – Weak acid production – Indication – S. aureus, E. faecalis

EMB Light#1 – Weak acid production – Indication – S. aureus, S. epidermidis, E. faecalis

Mac Light#1 – Weak acid production – Indication – S. aureus, S. epidermidis, E. faecalis

Reviewed Gram Negative agar plates.  Based on the Unknown Chart provided there were five possible Gram Negative Rod possibilities:  Escherichia coli, Klebsiella pneumonia, Enterobacter aerogenes,  Proteus vulgaris, Pseudomonas aeruginosa.  The test results were:

MSA Creamy#2 – Weak acid production – Indication – E. coli, K. pneumonia, E. aerogenes

EMB Creamy#2 – Weak acid production – Indication – E. coli, K. pneumonia, E. aerogenes   Growth was pink/purple colonies not metallic green which indicates E. coli – E. coli eliminated from possibilities

Mac Creamy#2 – Weak acid production – Indication – E. coli, K. pneumonia, E. aerogenes

Based on these results decided to run the following tests next lab period:

Gram Positive – Light#1 – Nitrate Test – differential media determines if an organism is capable of reducing Nitrate to Nitrite. Will differentiate between S. aureus and E. faecalis.

Gram Negative – Creamy#2 – Urea Test – used to determine is an organism is capable of hydrolyzing urea using the enzyme urease.  Will differentiate between K. pneumoniae and E. aerogenes

Gram Negative – Creamy#2 – Methyl Red Test – used to determine if an organism produces acid from glucose metabolism.  Will differentiate between K. pneumoniae and E. aerogenes.

 

Day 6 – November 7, 2013

The following media was inoculated according to procedures outlined in the lab manual:

Gram Positive Light#1 – Nitrate Test

Gram Negative Creamy#2 – Urea Test

Gram Negative Creamy#2 – Methyl Red Test

 

Day 7 – November 12, 2013

Reviewed tests ran during previous lab period and noted the following results:

Gram Positive – Light#1 – Nitrate Test – broth turned red after adding zinc – Negative result – Indication – Enterococcus faecalis

Gram Negative – Creamy#2 – Urea Test – no change in color – Negative result – Indication – Enterobacter aerogenes

Gram Negative – Creamy#2 – Methyl Red Test – yellow after methyl red was added – Negative result – No acid production – Indication – Enterobacter aerogenes

These results were reviewed.  Entercoccus faecalis was correct for Gram Positive.  Enterobacter aerogenes was incorrect for Gram Negative.

 

Day 8 – November 14, 2013

Gram stained sample from Gram Negative – Creamy#2.  Determined growth to be Gram Positive Rods. Determined that original streak of Unknown Sample #118 had been contaminated with unidentified bacteria.  Received sample ALT 5 – pure Gram Negative Bacteria of Unknown Sample #118. The following media were inoculated with ALT 5 according to procedures outlined in the lab manual:

ALT5 MAN – Mannitol Broth

ALT5 MAC – MacConkey Agar Plate

ALT5 MR – Methyl Red Broth

ALT5 UR – Urea Broth

ALT5 SC – Simmon’s Citrate Slant

Placed media in the incubator for 120 hours.

 

Day 9 – November 19, 2013

Reviewed tests performed on 11/14/13 with the following results:

ALT5 MAN – Mannitol Broth – Negative – No Acid production – Indication – Proteus vulgaris, Pseudomonas aeruginosa

ALT5 MAC – MacConkey Agar Plate – Negative – No acid production – Indication – Proteus vulgaris, Pseudomonas aeruginosa

ALT5 MR – Methyl Red Broth – Positive – Indication – Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris

ALT5 UR – Urea Broth – Negative – Indication – Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa

ALT5 SC – Simmon’s Citrate Slant – Negative – Indication – Escherichia coli, Proteus vulgaris

Though the results of the Urea test were conflicting with four other test the indication was the Gram Negative ALT5 was Proteus vulgaris.  This was incorrect.

Received ALT5 #2 – Pure culture of Gram Negative Unknown #118.




The following media were inoculated according to procedures outlined in the lab manual:

ALT52 MR – Methyl Red Broth

ALT52 LAC – Lactose Broth

ALT52 MAN – Mannitol Broth

ALT52 UR – Urea Broth

Placed in incubator for 48 hours.

 

Day 10 – November 21, 2013

Reviewed test ran on ALT5 #2 to identify Gram Negative Unknown.  The following results were noted:

ALT52 MR – Methyl Red Broth – Negative – Indication – Enterobacter aerogenes, Pseudomonas aeruginosa

ALT52 LAC – Lactose Broth – Negative – Indication – Proteus vulgaris, Pseudomonas aeruginosa

ALT52 MAN – Mannitol Broth – Negative – Indication – Proteus vulgaris, Pseudomonas aeruginosa

ALT52 UR – Urea Broth – Negative – Indication – Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa

These results pointed to Pseudomonas aeruginosa however this was incorrect for Unknown #118- ALT5#2.  Gram Negative for #118 was Klebsiella pneumoniae.  No test results indicated this bacteria.

Received ALT-1 a pure culture of a Gram Negative bacteria.  Performed the following tests on ALT-1.

 

Ran the following tests according to procedures outlined in the lab manual on ALT-1:

ALT1 MAN – Mannitol Broth

ALT1 LAC – Lactose Broth

ALT1 SC – Simmon’s Citrate Agar Slant

ALT1 MR – Methyl Red Broth

ALT1 UR – Urea Broth

ALT1 NT – Nitrate Broth

Placed in the incubator for 120 hours.

Day 11 – November 26, 2013

Reviewed test performed to identify Gram Negative of ALT-1.  The following results were noted:

ALT1 MAN – Mannitol Broth – Positive for acid production – Indication – Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes

ALT1 LAC – Lactose Broth – Positive for acid production – Indication – Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes

ALT1 SC – Simmon’s Citrate Agar Slant – Positive – Indication – Klebsiella pneumoniae, Enterobacter aerogenes, Pseudomonas aeruginosa

ALT1 MR – Methyl Red Broth – Negative – Indication – Pseudomonas aeruginosa, Enterobacter aerogenes

ALT1 UR – Urea Broth – Negative – Indication – Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa

ALT1 NT – Nitrate Broth – Positive – Indication – Klebsiella pneumoniae, Enterobacter aerogenes, Proteus vulgaris, Pseudomonas aeruginosa

Based on these results ALT-1 Gram Negative was Enterobacter aerogenes.  This was correct.

 

 

Results Summary

Gram Positive Unknown Pathway

  1. Gram Stain
  2. MSA Agar
  3. EMB Agar
  4. MacConkey Agar
  5. Nitrate Broth

Colonies on original streak of Unknown #118 were light white almost transparent in appearance.  Table 1 summarizes each test, purpose, materials and results for Gram Positive Unknown #118 pathway.

 

Gram Negative Unknown Pathway

  1. Gram Stain
  2. Mannitol Broth
  3. Lactose Broth
  4. Simmon’s Citrate Agar Slant
  5. Urea Broth
  6. Methyl Red Broth
  7. Nitrate Broth

Colonies were creamy in appearance. Table 2 summarizes each test, purpose, materials and results for Gram Negative ALT-1 pathway.

 

Table 1.  Gram Positive Unknown – Enterococcus faecalis

Test

Purpose

Main

Materials

Observations

Results

Gram StainTo determine the Gram reaction of the bacterium – Gram Positive or   Gram NegativeCrystal VioletGram Iodine

Gram Decolorizer

Gram Safranin

 

Purple CocciGram Positive Cocci
Mannitol Salt Agar TestTo determine the ability of a bacterium to ferment mannitol producing   acid Mannitol Salt Agar – phenol red PH indicatorSlight color change of agar to yellowAcid produced
Eosin – Methylene Blue AgarTo determine the ability of a bacterium to ferment lactose producing   acidAgar with Eosin and Methylene BlueVery light pink coloniesAcid produced
MacConkey AgarTo determine the ability of the bacterium to ferment lactoseAgar with bile salts and crystal violetLight pink purple coloniesAcid produced
NitrateTo determine the ability of the bacterium to reduce nitrate to   nitrite or other nitrogenous compoundsNitrate Broth tubes, Nitrate Reagent A, Nitrate Reagent B, ZincNo change after adding Reagent A and Reagent B. Turned red after   adding ZincNegative

 

 

 

Table 2.  Gram Negative Unknown – Enterobacter aerogenes

Test

Purpose

Main

Materials

Observations

Results

GramStainTo determine the Gram reaction of the bacterium – Gram Positive or   Gram NegativeCrystal VioletGram Iodine

Gram Decolorizer

Gram Safranin

 

Pink RodsGram NegativeRods
MannitolBrothTo determine the ability of a bacterium to ferment mannitol producing   acid Mannitol Broth – phenol red PH indicatorBroth changed from red to yellow indicating acid productionPositive for acid productions
LactoseBrothTo determine the ability of the bacterium to ferment lactose   producing acidLactose BrothBroth changed to yellow indicating acid productionPositive for acid production
SimmonsCitrateTo determine the ability of the bacterium to metabolize ammonium   phosphate – to use citrate as its sole carbon sourceCitrate agar slant – sodium citrate, ammonium phosphate, PH indicator   bromthymol blueAgar turned blue – PH > 7.6Positive for CO2 and pyruvic acid production
Methyl RedTo determine the ability of the bacterium to ferment glucose   producing acidBroth containing glucose, peptone and a PH buffer, PH indicator   methyl redBroth turned yellowNegative for glucose fermentation
UreaTo determine the ability of the bacterium to hydrolyze ureaUrea Broth, PH indicator phenol redYellow brothNegative for hydrolysis of urea, no ammonia production
NitrateTo determine the ability of the bacterium to reduce nitrate to   nitrite or other nitrogenous compoundsNitrate Broth tubes, Nitrate Reagent A, Nitrate Reagent B, ZincNo change in color after adding zinc to the broth tubePositive for nitrate reduction

 

 

 




Discussion / Conclusion

As noted in the Materials and Methods section of this report, the Gram Positive was identified after the first round of testing.  A pure Gram Positive colony was grown from the original streak of Unknown Sample #118, Gram Stained and basic testing lead to the correct identification of Enterococcus faecalis.

Also as noted in the Materials and Methods section, the identification of the Gram Negative bacteria of Unknown Sample #118 was incorrect multiple times.  A uniquely different colony was selected from the original streak of #118 and grown and Gram Stained as Gram Negative Rods.  However testing of this inoculated plate led to the conclusion that multiple species were growing on this plate.  Testing was performed on alternate versions of Unknown #118 Gram Negative but probable lab error during inoculation of media lead to incorrect results.  As noted, testing on a new alternate of a different species lead to a correct identification of a Gram Negative species Enterobacter aerogenes.

 

Scientific classification

Kingdom:  Bacteria

Phylum:  Proteobacteria

Class:  Gamma Proteobacteria

Order:  Enterobacteriales

Family:  Enterobacteriaceae

Genus:  Enterobacteriaceae

Species: Enterobacter aerogenes

 

Enterobacter aerogenes is a gram negative, facultative anaerobe rod whose name comes from the Greek enteron meaning “intestines” which is its natural habitat in the intestines of animals.  While it is generally found in the gastrointestinal tract, it has also been found in various wastes, hygienic chemicals and soil.    Often found in nosocomial environments it is an opportunistic pathogen that typically only infects those with a compromised immune system.  Surgical procedures, intravenous catheter insertions and some antibiotic treatments are common procedures that can introduce E. aerogenes that can lead to respiratory infections, urinary tract infections, osteomyelitis, septic arthritis and blood stream infections (3).  E. aerogenes is closely related to and often misidentified as Klebsiella pneumoniae however K. pneumoniae lacks Beta Lactamase.   In the mid 1980’s, E. aerogenes with Extended-Spectrum Beta-Lactamase were discovered meaning the bacterium was also capable of carbapenamase production.  This has made E. aerogenes infection epidemic and endemic worldwide (4).

 

Bibliography

  1. What is Bacteria? What are Bacteria? Last updated March, 2013.
  2. McDonald, V., Thoele, M., Salsgiver, B., Gero, S., Lab Manual for General Microbiology, Revised April 2011.
  3. www.britannica.com/EBchecked/topic/685512/Enterobacter
  4. www.biomedcentral.com/1471-2180/4/49

 

 

 

 

 

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