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Unknown Bacteria Lab Report | Microbiology Paper

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December 3, 2013



This study is the determination of the UNKNOWN bacterium that was given by Microbiology  LAB  Professor . The purpose of this laboratory exercise is to evaluate the student’s  knowledge and capabilities in identifying micro organism. Knowing what  organism will give the student an idea of the  genus  and species that can cause diseases, how it can grow and reproduce including how this type of bacteria can be treated and killed, what type of antibiotic it will be susceptible to.

This activity includes the application of procedure , technique  and skills related to what type of Agar to be use to grow culture, inoculating and sterilizing, reagents to apply, handling of samples away from contamination and the skills in gram staining , handling and use of microscope and the differentiate differential and selective test for gram-negative and gram-positive bacteria.



An unknown LABELED 105  was given by the Lab Professor.  From the knowledge acquired from laboratory exercise by Mc Donald, et al  (9), the following procedure and test that has been done are listed in enumerative form as follows;

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  • Culturing/Streak

The Nutrient Agar (NA ) plate was labeled : Student name and Unknown 105. Since the unknown sample contains a Gram (+ ) and a Gram (-) , it is just proper to make a growth for the unknown in an agar plate. Using a sterilized inoculating loop a sample was streak on the Nutrient Agar plate.  The purpose of this procedure is to allow growth of the UNKNOWN 105  in the plate.  Taking extra care to prevent contamination. The inoculated NA was incubated to  37 C for the next day.

  • Physical Examination of the NA

After 2 days, The NA plate was analyzed  through visual examination as to growth, distinct color and colony. There was growth on the NA plate and there was 2 distinct color which maybe a manifestation of 2 or more different types of bacteria.

  • Isolating a Pure Culture /Colony  (Mc Donald, et al p. 10 )

Since there were 2 distinct color and colony in the NA plate.  With a sterilized inoculating loop  the  colony was separately  inoculated to a new set of  NA for growth Taking extra care on label and streaking.  The purpose of this is to grow separately the 2 possible bacteria. It helps to isolate a possible a gram (+) and a gram (-). These 2 NA plate was incubated to 37 C to grow for next meeting.

  •  Gram Staining

After 2  days the 2 different NA plates  were examined and separately transferred into   the microscopic slide  to be examine  which is the Gram (-) and the gram (+) by using the methods Gram Staining (Tortora, et al. p.68-69)

Using a sterilized inoculating loop a drop distilled water in a dry glass slide which was previously soaked in alcohol.  The purpose of the water is to make a pool so that the Unknown specimen can be transferred and spread in the slide. The mixture of water and Unknown specimen A ) was air dried, then passed lightly  3 times in a heat so that the mixture will stick on the slide.  The mixture was soaked with Crystal violet for 30 sec.  The purpose of this is to stain the bacteria. After 30 sec the mixture was rinse with distilled water, taking extra care not to hit the mixture.  Then the mixture was soaked in Iodine for 1 min. The Iodine here acts as Mordant. Then washed with alcohol.  Visually it becomes colorless. A safranin was drop to the mixture to cover the colorless area and then washed again with distilled water.  The mixture was air dried and ready for Microscopic examination.


  • Microscopic Examination

The air dried slide was viewed first at 10X magnification, then to a 100X magnification with the immersion oil to cover the slide. The bacteria was colored  PINKEST RED  and the shape is ROD SHAPED  which are the characteristic features of a GRAM NEGATIVE BACTERIA.


  •  The save NA was labeled Gram Negative Bacteria was subject to the following test;
  •  Mannitol agar test
  • Casein test
  • Gelatin test
  • Indole
  • Simmon Citrate test
  • EMB
  • Urea
  • Catalase
  • Nitrate
  • Blood agar



Test Purpose Reagents Results
Gram Stain To determination gram reaction of the bacterium Crystal violet, distilled water, Iodine, Alcohol Safranin Pinkish Red color, Rod shaped.  The unknown  is gram (-) bacteria.
Mannitol To determine bacteria that grows in high salt MSA plate The Red agar plate becomes yellow color. The following bacteria are negative to mannitol; Bacillus cereus, Bacillus Subtilis, ,Staphylococcus Epidermidis.  The Staphylococcus  aureus and Enterococcus faecalis are positive to mannitol.
Milk (Casein) To determine if bacteria can breakdown casein and absorb amino acid on fermentation Casein Agar No Clearing on the side of the streak. No color change
Gelatin To determine if bacteria produce gelatinase on fermentation Gelatin Agar No growth, no change in color
Simmon Citrate To determine the ability of organism to detect enzyme Citrase Simmon Citrate Agar Change of Agar Color from Green to Blue.
Indole To determine the ability of the organism to split indole from tryptophan Sim tube, Indole reagent No Color Change
Urea To determine the ability of micro organism to detect Urease Urea broth, No change in color, no growth
Catalase To determine the ability of micro organism to detect presence of catalase Hydrogen Peroxide No bubbling. No reaction
EMB A selective test for Enteric bacteria that produces evident colonies Eosin  and methylene blue There’s growth of pink colonies. The Unknown bacteria is Enterobacter aerogenes



The other cultured  bacteria was gram stain using same procedure at Number 4 The second  streak culture was subjected to microscopic test.  In a 100x magnification  the color of bacteria was PURPLE and the shape is ROUND, GRAPE LIKE STRUCTURE. The following test were conducted.

Test Purpose Reagents Results
Gram Stain To determination gram reaction of the bacterium Crystal violet, distilled water, Iodine, Alcohol Safranin

Purple Color, round, grapelike structure. Indication of gram (+) bacteria. My unknown can either be any of the following gram (+) bacteria ; Bacillus Cereus, Bacillus  Subtilis, Stapahylococcus Aureus, Staphylococcus Epidermidis or Enterococcus faecalisMannitolTo determine bacteria that grows in high salt concentrationMannitol agarRed agar mannitol turns yellow colorUreaTo determine if bacteria can detect ureaseUrea brothNo change in color.  The Unknown is negative to ureaCatalaseTo determine the ability of micro organism to detect presence of catalaseHydrogen PeroxideBubbling . The bacteria  detect enzymes amylase. Amylase is the enzymes that chopped up the Hydrogen Peroxide to water and Oxygen .NitratesTo determine the ability of bacteria to convert NO3 to NO2Nitrate broth, Reagent A , Reagent BThere’s no change in color. The bacteria did not detect  a change of  Nitrate to Nitrate even with the addition of Zn.Blood AgarThe ability of bacteria to clump and produce hemolysin

Sheep blood agar

The red agar plate has shown growth and partial clearing on the side of the streak like a ring. The unknown bacteria is Staphylococcus aureus.



Culture NA 37C Growth of Unknown Sample 2 distinct color and colonies This means the Unknown sample has 2 or more different kinds of bacteria
Gram Stain Crystal violet, Iodine, Alcohol, safranin, distilled water microscope, oil Room temp

25 CThe Crystal violet colors / stain the gram positive.  The Iodine was used as mordant and become colorless when alcohol was used. Safranin was used to color the colorless bacteria. The gram (+) is purple.  The gram(-) is redPositive of gram(-) bacteria.

The other plate has gram (+) bacteriaThe gram-negative is pinkish red with a rod shaped , cocci.

The  gram positive bacteria is colored purple with a grape-like structure, a StaphylococcusMannitolMSA plate35CThe Red Agar plate becomes yellow after 2 days. There’s a yellowish zone aroundPositiveGram (+) organism turn Agar red to yellow indicates growth of gram positive bacteria. The bacteria ferments mannitol. A differential medium for   mannitol fermentors like S.Aureus and E. faecalis  produce yellow colonies with yellow zonesMilk CaseinCasein Agar 35 CThe color of Casein agar was the same. Light peach to peach, No Clearing on the side of the streak.NegativeGram (-) Organism does not produce enzyme casein. The organism does not ferment in milkGelatinGelatinase35 CThe color of Gelatin agar was the same. No clearing on the side of the streakNegativeGram (-) Organism does not produce gelatinase. Does not ferment in gelatine.Simmon CitrateCitrate Slant (green)35 CAfter 2 days the Green Citrate Agar slant becomes bluePositiveGram (-)Organism is able to utilize enzyme citrate as a source of Carbon . The bacteria that produce the enzymes citrase breakdown the citrate, changing the PH of the agar slant and shifting its color from green to blue. Manifestation of a gram negative bacteriaIndoleSim tube, Indole  Rgt35 CNo Color ChangeNegativeGram (-)did not split the indole  from tryptophanUreaUrea broth35 CThe urea test tube is light peach color, after 2 days there was no change in colorNegativeThe Gram (-) like E.coli and E. Aerogenes organism does not produce Urease.

Gram (+) organism did not have any change in color at the Urea tube.EMBEMB agar plate35 CThere are colonies that are pink dark center like a wide mucoid rim coloniesPositive The gram-negative bacteria reacts to the eosin blue. This type of colony are methyl red-negative lactose-fermenters.CatalaseH2O225CAdding  a gram positive bacteria to the pool of hydrogen peroxide in the slide produce bubbles.

Gram (+) bacteria produce bubbles.PositiveBubbling means the  break down of Hydrogen Peroxide into water and Oxygen. The O2 is the bubbling reaction.     The organism detects the presence enzyme   catalase.NitratesNitrate broth, Reagent A , Reagent B, Zn35 CTo determine the ability of bacteria to convert NO3 to NO2Positive Theres no change in color. The bacteria did not detect  a change of  Nitrate to Nitrate even with the addition of Zn.Blood AgarSheep blood

35 CThe red agar plate has shown growth and clearing on the side of the streak.PositiveThe bacteria produces a partial hemolysis as manifested by the ring around the streak. The bacteria  released an alpha-toxin. The bacteria is Staphylococcus Aureus.



Gram Stain


       Microscopic                                                                          Microscopic

(Pinkish/red color, Rod shaped cocci                       (Purple color, round, grape like structure )

      GRAM NEGATIVE )                                                    GRAM POSITIVE

Escherichia coli                                                                   Bacillus cereus

     Kleibsiella pneumoniae                                                        Bacillus  subtilis

     Enterobacter aerogenes                                                        Staphylococcus aureus

     Proteus vulgaris                                                                   Staphylococcus epidermidis

     Pseudomonas Aeruginosa                                                    Enterococcus faecalis


Mannitol test                                                                       Mannitol test


Negative                            Positive                                              Negative                   Positive     

Proteus vulgaris                 Escherichia coli                               Bacillus cereus           S. aureus 

Pseudomonas Aeruginosa   Kleibsiella pneumoniae                 Bacillus  subtilis         E.faecalis

Enterobacter aerogenes                   S. epidermidis

CASEIN/GELATIVE                                                           CATALASE

                Negative                                                                       Negative                Positive                

Escherichia coli                                                     E.faecalis                 Bacillus cereus

Kleibsiella pneumoniae                                         Bacillus  subtilis

Enterobacter aerogenes                                         Staphylococcus aureus

S. epidermidis



          Negative                                   Positive                                                  UREA

    Kleibsiella pneumoniae          Escherichia coli                             Negative                   Positive

Enterobacter aerogenes                                                           Bacillus cereus           S. epidermidis

Bacillus  subtilis

SIMMON CITRATE(positive)                                          NITRATE ( Positive)                                                   

              Enterobacter aerogenes                                                        Staphylococcus aureus


EMB (positive)                                                                BLOOD AGAR( positive)        

Enterobacter aerogenes                                                      Staphylococcus aureus   


UNKNOWN  : Enterobacter aerogenes                        UNKNOWN:  Staphylococcus aureus

NEGATIVE                                                                     POSITIVE



The Unknown Sample which is a combination of a gram (-) and a gram(+) was cultured into a Nutrient Agar plate and incubate to 35 C.  After 2 days, the growth was examine as to the morphology , color and colony.  Evident colonies with distinctively color were isolated into  2 separate culture   plate. Since there were 2 distinct color and colony in the NA plate.  With a sterilized inoculating loop  the  colony was separately  streak , inoculated and incubated. The purpose of this is to grow separately the 2 possible bacteria. It helps to isolate a possible a gram (+) and a gram (-). These 2 NA plate was incubated to 35 C to grow for next meeting.

Next  day, a gram stain was done as explained on Number 4 above. The slide was viewed on an oil immersion 100x.  The bacteria was shown as rod shaped  type cocci pinkish color which is a characteristic feature of a gram negative bacteria.  The plate with the gram-negative is labeled and keep for future test.

The other NA growth was then gram stained  and viewed into an immersion 100x magnification and it showed a purple color, grape-like structure prominent of a cluster staphylococcus , a  gram positive bacteria.  The plate was labeled gram positive for future test.

The gram negative bacteria was done through elimination process following the Unknown Chart given by the Lab Professor. The bacteria was tested on the mannitol test and turns read mannitol to yellow.  This eleminates the 2 bacteria leaving Escherichia coli, Klebseilla pneumoniae, Enterobacter aerogenes and Proteus vulgaris ) The  3 gram negative bacteria was tested using the gelatine, milk, urea and indole test simultaneously to find out if the gram-negative bacteria can detect enzymes casein, gelatinase , urease and indole. The bacteria did not split indole from tryptophan.  The result are negative to the 3 tests.  The bacteria was subjected to Simmon Citrate test.  The Original color of simmons is green. Incubated for 2 days.After 2 days the green simmon citrate turns to blue. Gram (-)Organism is able to utilize enzyme citrate as a source of Carbon, manifestation of a gram negative bacteria. Looking at the unknown chart this eliminates E. coli leaving 2  gram negative bacteria ( K. pneumoniae and E. aerogenes ) .  The bacteria was subjected to  EMB which shows pink colonies indicative of a  gram-negative Enterobacter aerogenes which reacts to eosin dye and negative to methyl red lactose fermenters.  The unknown gram-negative bacteria is Enterobacter aerogenes.

The other bacteria which was microscopically viewed as gram positive was missing in the incubator, another tube of sample was  derived from the lab professor. The gram positive bacteria was subjected to the following test; First the gram (+) bacteria was tested on catalase test. The bacteria produces bubbles with Hydrogen Peroxide . This eliminates Entercoccus faecalis bacteria leaving  4 which are Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis. These bacateria where subjected to urea test which eliminates the S. epidermidis. These bacteria was sbjested to Nitrate test which eliminates the  B. subtilis and B. cereus leaving S. aureus for final testing.  The bacteria was streak in a blood agar and it produces an alpha-toxin hemolysis showing a partial clearing on the side of the streak.  The unknown gram positive bacteria is Staphylococcus aureus.

Some problems encountered on this activity is the handling and contamination. Due to a lot of plate and tubes in one incubator, some samples are missing.




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