Laboratory Report

Identification of Unknown Bacteria from Environmental Isolates

Introduction:

Isolation and identification of bacteria from the human body or the environment are crucial for the prevention and treatment of disease. Bacteria can be found throughout the world, with their composition depending on the system that exist in. The human body has a wide variety of normal flora, with different regions having different species prevalent. The transposition of bacteria from one region to another can result in the bacteria becoming an opportunistic pathogen, causing disease and harm to the individual (2). 

Colony morphology, Gram staining and biochemical tests provide important information that enable identification of unknown isolates. Gram staining utilizes differences in cell composition between two types of bacteria to facilitate preliminary identification and visualization of cell structure. Gram positive bacteria are commonly found on the skin and in the oral cavity of humans, while Gram negative bacteria are normally associated with the digestive tract. The presence or absence of certain Gram negative bacteria, called coliforms, are used to determine the safety of water for human use, with their presence indicating fecal contamination (3).

Methods (3): 

Isolation of device bacteria:

• Using a sterile swab moistened with sterile saline, the back of a classmate’s Apple Watch was swabbed with particular attention to the area where the band attaches.
• The swab was gently rolled along the surface of a blood agar plate near the edge. 
• With a sterile inoculating loop, the sample was streaked for isolation using the T-streak method. 
• The plate was incubated in a candle jar at 37°C for 24 hours, then observed for growth. 

Dental carries testing:

• Saliva was added to a tube containing molten Snyder agar, then gently swirled to induce mixing before the agar cooled and solidified. 
• The tube was capped and incubated at 37°C for 24 hours, then observed for growth and media color change.

Isolation of water bacteria:

• A serial dilution of water sample #3 was performed by transferring 0.5mL of the sample into 4.5mL of sterile saline water to create the following dilutions: 10⁰, 10^-1, and 10^-2. 
• 0.1mL of each dilution was applied to 3 MacConkey agar plates for a total of 9 plates. 
• A glass spreader, sterilized using ethanol and a bunsen burner, was used to evenly distribute the water sample across the surface of the plate while being rotated on a turntable. 
• The plates were incubated at 37°C for 24 hours, then observed for growth and colony counts. 

Gram-staining of isolates:

• A prominent yellow colony from the BAP was selected for further study. 
• Due to uniform colony formation, an isolated colony from one 10^-2 plates was selected for further study as a representative of the entire plate.
• A drop of deionized water was applied to individual slides.
• Using a sterile loop, a small amount of bacteria from the yellow colony was transferred to one slide. The process was repeated with a sterile loop for the spread plate colony. 
• The slides were dried on a slide warmer and remained for an additional five minutes to ensure the samples had been heat-fixed.
• Crystal violet was applied to the sample on each slide for 1 minute, then rinsed off using DI water. 
• Each slide was flooded with Gram’s iodine for 1 minute, then rinsed using DI water. 
• With the slide held at an angle, acetone was used to rinse the slide for approximately 10 seconds, until no more crystal violet ran out, then immediately rinsed with DI water. 
• Safranin was applied to each slide for 45 seconds then rinsed with DI water.
• Each slide was gently blotted on bibulous paper, then observed with the 100x objective using immersion oil.
• After completion of Gram stain, each isolate was subcultured to individual tryptic soy agar plates and incubated at 37°C for 24 hours. 

Biochemical testing of isolates:

• Catalase test:
  • Using a sterile loop, a small amount of the device isolate from the TSA plate was smeared onto a glass slide. 
  • A drop of hydrogen peroxide was applied to the smear and observed for reaction.
• Oxidase test:
  • Oxidase reagent was applied to filter paper until saturated. 
  • Using a sterile swab, a small sample of the water isolate was applied to a section of the filter paper and observed for a reaction within 30 seconds.
• Coagulase test:
  • A small sample of the device isolate was transferred into the coagulase tube containing 0.5mL of diluted rabbit plasma using a sterile loop.
  • The tube was capped and incubated at 37°C for 24 hours, then observed for changes to the media.
• Hemolysis test:
  • A sample of the device isolate was streaked onto a BAP using a sterile loop. 
  • The plate was incubated in a candle car at 37°C for 24 hours then observed for hemolysis.
• Starch hydrolysis test:
  • Using a sterile loop, the device isolate was streaked in a center line down the starch plate and incubated at 37°C for 24 hours. 
  • After incubation, the plate was observed for growth then flooded with iodine and observed for clearing around the colony. 
• Antibiotic susceptibility tests:
  • A sample of the device isolate was streaked onto 2 BAPs and 1 TSA. 
  • A bacitracin disk was applied centrally to one BAP using sterile forceps. An optochin disk was applied to the other BAP. A furazolidone disk was applied to the TSA.
  • All plates were incubated at 37°C, with the BAPs in a candle jar, for 24 hours then observed for clearing around the disks.
• Esculin hydrolysis test:
  • The bile esculin slant was inoculated with a sample of the device isolate using a sterile inoculating needle to stab the media, then a sterile loop to streak along the slant surface. 
  • The tube was capped and incubated at 37°C for 24 hours, then observed for growth and color changes in the media.
• Urease test:
  • Using a sterile loop, each urea tube was inoculated with one of the isolates in a fishtail streak. 
  • The tubes were capped and incubated at 37°C for 24 hours then observed for growth and media color changes. 
• Nitrate reduction test:
  • Each nitrate broth tube was inoculated with one of the isolates using a sterile loop, then capped and incubated at 37°C for 24 hours.
  • After incubation, tubes were observed for growth.
  • Four drops of Solution A and 4 drops of Solution B were added to each tube, then observed for color change.
  • If no color change occurred, zinc dust was added to the tube and observed for color change.
• Methyl red and Voges-Proskaur tests:
  • Using a sterile loop, 2 MRVP tubes were inoculated with the device isolate and another 2 with the water isolate. 
  • All tubes were capped and incubated at 37°C for 24 hours. 
  • After incubation, 5 drops of methyl red pH indicator was added to one tube of each organism and observed for color change.
  • To the remaining tubes, 30 drops of ⍺-naphthol was added then the tubes were gently swirled to induce mixing.
  • 10 drops of 40% potassium hydroxide solution was added to each tube and the tubes were swirled again.
  • The tubes were incubated in a slanted position at room temperature for 60 minutes, then observed for color change.
• Carbohydrate fermentation tests:
  • A sample of the device isolate and water isolate were transferred to individual glucose fermentation tubes using a sterile loop.
  • The mannitol fermentation tube was inoculated with the device isolate using a sterile loop.
  • A sample of the water isolate was transferred to the lactose and sucrose fermentation tubes using a sterile loop. 
  • All tubes were capped and incubated at 37°C for 24 hours, then observed for color changes to the media and evidence of gas production.
• Rapid lactose fermentation test:
  • Using a sterile loop, an isolation streak of the water isolate was applied to the eosin methylene blue plate.
  • The plate was incubated at 37°C for 24 hours, then observed for growth and colony morphology.
• Citrate utilization test:
  • The citrate tube was inoculated with a sample of the water isolate using a sterile loop and fishtail streak on the slant surface. 
  • The tube was capped and incubated at 37°C for 24 hours, the observed for growth and media color changes. 
• Triple-sugar iron test:
  • The triple-sugar iron slant was inoculated with a sample of the water isolate using a sterile needle to stab the media, then a sterile loop to streak the slant surface. 
  • The tube was recapped and incubated at 37°C for 24 hours, then observed for color change in the media and cracking or lifting. 
• O/F glucose test:
  • Each O/F glucose tube was inoculated with a sample of the water isolate using a sterile needle. 
  • 10 drops of mineral oil were layered at the top of one tube.
  • Both tubes were recapped and incubated at 37°C for 24 hours, then observed for growth and color changes in the media.
• Phenylalanine deaminase test:
  • The phenylalanine slant was heavily inoculated with the water isolate using a sterile loop to create a fishtail streak.
  • The tube was recapped and incubated at 37°C for 24 hours.
  • After incubation, 5 drops of 10% ferric chloride solution was added to the tube and immediately observed for reagent color change. 
• Tryptophan hydrolysis test:
  • The tryptophan broth was inoculated with a sample of the water isolate using a sterile loop. 
  • The tube was recapped and incubated at 37°C for 24 hours. 
  • After incubation, 12 drops of Kovac’s reagent was added to the tube and observed immediately for reagent color change.
• Decarboxylase tests:
  • The lysine, ornithine, and decarboxylase base tubes were each inoculated with a sample of the water isolate.
  • The tubes were recapped, then incubated at 37°C for 24 hours. 
  • After incubation, tubes were observed for media color.

Results:

Test	Growth	Result	Observations
Gram stain		+ve	Purple cocci in clusters, tetrads and pairs
Colony morphology	+ve		Small, round, entire, yellow colonies
Catalase test		+ve	Bubbles
Coagulase tube	-ve	-ve	No clumping observed
Hemolysis plate	+ve	ɣ	Green halo around colonies
Starch hydrolysis plate	+ve	-ve	No clearing around colonies
Bacitracin susceptibility plate	+ve	Susceptible	Clearing around disk
Optochin susceptibility plate	+ve	Resistant	No clearing around disk
Furazolidone susceptibility plate	+ve	Resistant	No clearing around disk
Urea slant	+ve	-ve	No color change to media
Bile esculin slant	-ve	-ve	No growth
Nitrate broth tube	+ve	-ve	Broth turned red after addition of zinc dust
Glucose fermentation tube	+ve	-ve	No color change to media
Mannitol fermentation tube	+ve	-ve	No color change to media
Methyl red tube	+ve	-ve	No color change to reagent
Voges-Proskauer tube	+ve	-ve	No color change to reagent

Test	Growth	Result	Observations
Gram stain		-ve	Elongated pink bacilli
Oxidase test		-ve	No color change
MacConkey plate	+ve	+ve	Round, mucoid, fuschia colonies
Eosin methylene blue plate	+ve	+ve	Round, mucoid, metallic green colonies
Simmon’s citrate slant	+ve	-ve	No color change to media
Urea slant	+ve	+ve	Media turned bright pink
Triple sugar iron slant	+ve	A/A + gas	Yellow butt and slant with cracks
Phenylalanine slant	+ve	-ve	No color change to reagent
Indole broth tube	+ve	-ve	No color change to reagent
Nitrate broth tube	+ve	+ve	Color change with addition of solutions A&B
Glucose fermentation tube	+ve	+ve	Yellow broth with gas in Durham tube
Sucrose fermentation tube	+ve	+ve	Yellow broth
Lactose fermentation tube	+ve	+ve	Yellow broth
O/F Glucose tube (aerobic)	+ve	+ve	Yellow broth
O/F Glucose tube (anaerobic)	+ve	+ve	Yellow broth
Base decarboxylase tube	+ve	-ve	Yellow broth
Lysine decarboxylase tube	+ve	+ve	Dark purple broth
Ornithine decarboxylase tube	+ve	-ve	Yellow broth
Methyl red tube	+ve	+ve	Reagent turned red
Voges-Proskauer tube	+ve	-ve	No color change to reagent

Discussion:

Based on colony morphology, the Gram stain, and biochemical test results, the bacteria isolated from the Apple Watch was identified as Micrococcus luteus. The round, yellow colonies on tryptic soy agar are a distinct feature of M. luteus, which is a member of the normal skin flora. Additionally, the purple cocci in tetrads and clusters were indicative of M. luteus (4). Further biochemical testing confirmed this identification.

The application of hydrogen peroxide resulted in bubbling, indicating the presence of catalase that causes the hydrogen peroxide to break down into water and oxygen. The formation of oxygen creates the bubbles, so this organism is catalase positive. Differentiating it from Staphylococcus aureus, another catalase positive bacteria commonly found on skin that produces golden colonies, is possible through the coagulase test. The production of coagulase by some bacteria induces the conversion of fibrinogen to fibrin, creating a clot. There was no evidence of clotting in the rabbit plasma, indicating the bacterial isolate is coagulase negative. S. aureus is positive for coagulase, so it can be ruled out as the potential species isolated from the Apple Watch. In addition, S. aureus is β-hemolytic while the isolated ⍺-hemolytic. M. luteus is both coagulase negative and ⍺-hemolytic, further confirmation that the isolate had been appropriately identified (3). 

Other species of Staphylococcus are coagulase negative and can be ⍺-hemolytic. To differentiate M. luteus from these possibilities, a furazolidone susceptibility test was used. M. luteus is resistant to furazolidone, which is seen as a lack of clearing around the disk. The isolate grew up to the edge of the disk, with no clearing observed, indicating it is resistant to furazolidone and not a species of Staphylococcus. It was also observed that the isolate was resistant to optochin, but sensitive to bacitracin based on the clearing seen around the disk. This indicates bacitracin would be a potential treatment for an opportunistic infection caused by M. luteus (3).

The following tests were negative and supported the identification made based on the above results. There was no clearing observed around the colony after the addition of iodine to the starch plate, This indicated the presence of starch throughout the media and that no amylase was produced by the bacteria to cause the starch to break down. There was no color change observed in the urea slant media despite growth being present, indicating the isolate does not produce urease that would break down urea in ammonia. The bile esculin slant did not have any growth, indicating the isolate is not bile tolerant and confirming it is not an Enterococcus species. The lack of color change in both the glucose and mannitol fermentation tubes despite growth observed in the tube indicates that the isolate does not ferment sugars, which would have resulted in a lowered pH that caused the media to turn yellow due to the phenol red pH indicator. Both the methyl red and Voges-Proskauer tests were negative, evidenced by the lack of color change after the addition of the reagents, indicating that neither mixed acid fermentation or pyruvate fermentation had occurred. This confirms that the isolate is not fermentative. The nitrate broth showed no color change after the addition of solutions A and B indicating no nitrite was present, but did change color after the addition of zinc dust. The zinc dust causes the reduction of nitrate to nitrite and confirms that the nitrate had not been reduced by the bacterial isolate (3).

The results of the biochemical tests serve to confirm the initial presumptive identification based on the colony morphology and Gram stain. One additional test that could be done to further support identification of the bacteria isolated from the Apple Watch would be a modified oxidase, or microdase, test. M. luteus is oxidase positive, meaning that it produces oxidase to catalyze the reduction of cytochrome c as part of the electron transport chain. Staphylococcus species do not produce oxidase, and therefore produce a negative result for this test, while M. luteus does (4).

The bacteria isolated from water sample #3 was identified as Klebsiella pneumoniae based on the colony morphology, Gram stain, and biochemical tests. The mucoid, lactose-fermenting colonies seen on the MacConkey agar are distinct to Klebsiellaspecies (1). It is a coliform bacteria that can be found in the intestines of animals, including humans, that indicates the presence of fecal contamination in water systems. It is an opportunistic pathogenic capable of causing pneumonia, as its name indicates, generally in immunocompromised patients (4).

The Gram stain showed elongated bacilli, occasionally in pairs, with slightly pointed ends. There was no color change during the oxidase test, indicating the bacteria isolated from the water sample does not produce the oxidase enzyme. Along with the fuchsia colonies on the MacConkey agar, these results indicate the isolate is a member of Enterobacteriaceae. This was further confirmed by the colony morphology seen on the eosin methylene blue plate. Rapid fermentation of lactose resulted in metallic green mucoid colonies from the reactions of the dyes with the acid products from fermentation (1).

In order to determine the genus and species if the isolate, further biochemical testing was required specific to members of Enterobacteriaceae. The triple-sugar iron slant utilizes phenol red as a pH indicator to determine acid production through fermentation both aerobically and anaerobically. It also tests for the ability of the bacteria to reduce sodium thiosulfate to hydrogen sulfide, which binds with the iron present in the media to produce a black precipitate. Both the slant and butt of the TSI were yellow, indicating fermentation throughout. Additionally, the cracks and lifting of the agar indicate the production of gas during fermentation. The carbohydrate fermentation tubes also indicated the organism ferments glucose, lactose and sucrose based on the production of acid turning the phenol red pH indicator in the media yellow. The O/F glucose test confirmed that the organism is a fermentor, producing acid through fermentation in both the aerobic and anaerobic tubes resulting in the lower pH turning the bromothymol blue pH indicator turning yellow (3).

In addition to fermentation, several other biochemical tests were conducted. The presence of urease produced by the bacteria was detected by the color of the urea slant changing from a pale peach color to bright pink. This occurred due to the breakdown of urea into ammonia which increased the pH and turned the phenol red pH indicator pink. The bacterial isolate was found to be capable of nitrate reduction based on the color change of the broth from pale yellow to red with the addition of solutions A and B. This indicates the organism is able to utilize nitrate as a terminal electron acceptor in place of oxygen during anaerobic respiration, causing it to be reduced to nitrite that is detected by the reagents. The isolate also produces lysine decarboxylase for amino acid metabolism, which breaks down lysine. This was determined by the purple color of the lysine decarboxylase tube in contrast to the yellow decarboxylase base tube. Initially, all tubes turned yellow due to acid production from fermentation. In the presence of lysine, production of lysine decarboxylase is induced and utilized in amino acid metabolism which creates basic conditions that return the pH indicator to purple. The positive methyl red test indicates mixed acid fermentation with the pH indicator methyl red, which is red in the presence of strong acids. The negative Voges-Proskauer test indicates the organism did not produce acetoin as a product of pyruvic acid metabolism (3).

The isolated organism was negative for both indole production and phenylalanine deaminase. The indole test determines whether the organism can break down tryptophan into indole using Kovac’s reagent. If indole production has occurred, the reagent turns red, but no color change was observed. This is important in distinguishing the isolate from Escherichia coli, another common Gram negative bacteria associated with water contamination. The production of phenylalanine deaminase, and subsequently the ability to metabolize phenylalanine, is indicated by a brief reagent color change from yellow to green from the reaction with ferric chloride. No color changed occurred with this organism The Simmon’s citrate slant tests for citrate utilization as a carbon source by bacteria, indicated by a color change of the media from teal to deep blue. This organism did not utilize citrate based on the lack of color change, which is an unexpected result. Citrate utilization requires aerobic conditions and it’s possible the cap had been applied too tightly to facilitate this (3). 

Despite the inconsistent citrate result, the above tests confirmed that the organism is Klebsiella pneumoniae. As a coliform, its presence would not be unexpected in a contaminated water sample and indicates the water is not safe for consumption or recreation. This could be further confirmed by performing an API test system for the sample to match against the index profile (3).

While growth was observed in the Snyder agar, it remained green. This indicates the bacteria present do not produce acid through the fermentation of glucose, which would decrease the susceptibility to dental caries. Caries form from the breakdown of dentin by acid, either from diet or from production by bacteria. Certain bacteria will ferment glucose available from the diet and produce acid as a byproduct that accelerates the formation of caries. Adequate oral hygiene should be sufficient to prevent the occurrence of caries in this situation (3).

Conclusions:

Analysis of the colony morphology, Gram stain and biochemical test results allows for identification of the bacterial isolates to be made with a high level of confidence. Micrococcus luteus is a common resident of the skin and could reasonably be expected on the inside band of a watch. The presence of Klebsiella pneumoniae in the water sample indicates contamination of the water source that may be unsafe for humans, but is not an unexpected indicator of this. 

References:

(1) Leboffe, M. J. and Pierce, B. E. (2005). Photographic atlas for the microbiology laboratory (3rd edition). Morton Publishing Company. 

(2) Madigan, M. T., Bender, K. S., Buckley, D. H., Sattley, W. M., Stahl, D. A. (2017). Brock biology of microorganisms (15th edition). Pearson. 

(3) Microbiology Lab Manual, 2019 

(4) Tortora, G. J., Funke, B. R., and Case, C. L. (2013). Microbiology: An introduction (11th edition). Pearson.