UNKNOWN LAB REPORT
DAISY M. BLACK
December 3, 2013
Introduction
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.
MATERIALS AND METHODS
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;
- 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
RESULTS : A. TEST GRAM NEGATIVE
| 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 |
B. TEST GRAM POSITIVE
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.
TABLE 3. PHYIOLOGICAL AND BIOCHEMICAL RESULTS
| TEST | REAGENTS OR MEDIA | TEMP | OBSERVATIONS | RESULTS | INTERPRETATIONS |
| 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.
FLOWCHART
UNKNOWN 105
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
INDOLE
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
DISCUSSION/CONCLUSION
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.
- Mc Donald, M. Thoele, S. Gero, Lab Manual for General Microbilogy, St Louis Community College, Meramec, April 2011
- Tortora G, Berdell F, North Dacuta State University, Boston,11th ed.2013
- http://en.wikipedia.org/wiki/Enterobacter_aerogeneHYPERLINK “http://en.wikipedia.org/wiki/Enterobacter_aerogenes”s
- http://www.ncbi.nlm.nih.gov/books/NBK8448/
- http://en.wikipedia.org/wiki/Staphylococcus_aureus
- http://www.medicinenet.com/staph_infection/article.htm
