An Outline of Identification Techniques of Unknown Bacteria

Introduction

The identification of bacteria, or what Steel refers to as the practical application of taxonic knowledge, is vital and pertinent to an array of fields (Steel 1965). Identifying bacteria can aid epidemiologists in tracing the sources of infections and addressing these sources to prevent further infections from occurring (Nungester 1963). It allows for the assessment of microbial diversity and the monitoring of food purity (Madigan et al. 2015). Identifying bacteria also has medical implications: it allows for the accumulation of data of interest to the study of infectious disease, determines an infection’s susceptibility to antimicrobial drugs, provides diagnostic information to physicians, and identifies pathogens with respect to their potential danger to patients and those in close contact with them (Nungester 1963).

There are a variety of techniques and tests available to aid in the process of identifying bacteria. New molecular technologies in genomic and proteomics are beginning to replace more traditional methods of bacterial identification, characterization, and classification (Emerson et al. 2008). These techniques rely on exploring specific gene sequences or molecular aspects of the unknown bacteria (Emerson et al. 2008). Genotypic methods of identifying bacteria include pattern or fingerprint based techniques (such as repetitive element polymerase chain reaction or rep-PCR, amplified fragment length polymorphism, riboprinting, random amplification of polymorphic DNA, pulsed-field gel electrophoresis, and multiplex PCR) and sequenced based techniques (such as small-subunit ribosomal gene sequencing, or SSU rDNA, multilocus sequence typing, or MLST, and multilocus sequence typing) (Emerson et al. 2008).

With this in mind, I set out to identify unknown organism 30.

Materials and Methods

Obtaining pure culture

To isolate single colonies, a loopful of a fresh broth culture of the unknown organism was streaked onto a nutrient agar plate and incubated overnight at 37˚C (Identification and report on unknown bacterial culture).

Microscopy

When required, microscopy was conducted using the 100x magnification objective of a confocal microscope.

Overnight cultures

Unless otherwise stated, the media was inoculated using a fresh, pure culture that was typically 18-24 hours old.

Incubation of samples

Unless otherwise stated, all samples were incubated at 37˚C with or without aeration.

Morphology characterization

Gram staining

To verify a Gram reaction, a Gram staining was performed following standard protocol and using a fresh broth culture (less than 24 hours old).

Acid fast staining

To verify the identity of acid-fast organisms, a Ziehl-Neelsen stain was performed using a fresh broth culture (less than 24 hours old) of the unknown organism. Carbolfuschin was used as the primary dye, and Methyl blue as the secondary dye. Samples were allowed to air dry.

Capsule staining

To verify the presence of a capsule, a capsule stain was performed using a fresh broth culture (less than 24 hours old) of the unknown organism. 1% Congo red was used as the primary dye, and Maneval’s stain as the secondary dye. Samples were allowed to air dry.

Cultural Characterization

Growth in solid media

To observe growth in solid media, a loopful of a fresh culture was inoculated on a nutrient agar slant and incubated overnight at 45˚C without aeration.

Oxygen requirements

To characterize the oxygen requirements, a fresh culture was inoculated in a tube of thioglycollate media using a single stab. Sample was incubated as mentioned above without aeration.

Biochemical and Physiological Characterization

Catalase test

To test for the presence of the catalase enzyme, a catalase test was performed using 3-5 colonies from a fresh agar plate (less than 48 hours old) of the unknown organism. A colony paste was made using sterile water on top of a microscope slide, and 1-2 drops of hydrogen peroxide were added. Results were observed within a minute.

SIM test

To test for the ability to produce sulfide, the ability to form indole, and motility, a SIM test was performed. Isolated colonies from a fresh agar plate (less than 48 hours old) were inoculated in the SIM media by stabbing into the center of the agar to a depth of ½ inch. The tube was incubated at 37˚C for 18-24 hours with aeration. After sulfide production and motility were observed, 3 drops of Kovac’s reagent were applied to the surface of the media.

Triple sugar iron agar test

To test for the ability to ferment sugars and produce hydrogen sulfide, a triple sugar iron (TSI) agar test was performed. A fresh broth culture was used to heavily inoculate the slant, then stab almost to the bottom of the agar butt, and streak the top of the slant. The tube was incubated at 37˚C for 16-24 hours.

Oxidase test

To test for the presence of certain cytochrome c oxidases, such as cytochrome oxidase or indophenol oxidase, a swab-based oxidase test was performed. The sterile oxidase test swab was dipped in sterile water then rolled on isolated colonies on a fresh agar plate. Results were observed within 20 seconds.

Nitrate reduction test

To test for the presence of the nitrate reductase enzyme and the nitrite reductase enzyme, a nitrate reduction test was performed. A loopful of fresh broth culture was used to inoculate the tube. The tube was incubated at 37˚C for 16-24 hours. After this time, a dropperful of sulfanilic acid and α-naphthylamine were added.

Phenylalanine deaminase test

To test for the presence of the deaminase enzyme, a phenylalanine deaminase test was performed. A fresh broth culture was used to heavily inoculate the slant. The tube was incubated at 37˚C for 16-24 hours. After this time, drops of 10% ferric chloride were added to the slant.

MR-VP (methyl red Voges-Proskauer) test

To determine if the unknown organism utilizes the mixed acid fermentation pathway, a MR (methyl red) test was performed. A 3-day (72 hours) old broth culture was used, to which 5 drops of methyl red were added. Results were observed within a few minutes.

To determine if the unknown organism utilized the butylene glycol pathway to produce acetoin, a VP (Voges-Proskauer) test was performed. A test tube with 1 mL of fresh broth culture had 0.6 mL α-napthol solution and 0.2 mL potassium hydroxide solution added. The tube was shaken gently and allowed to stand for 5-10 minutes. Results were observed within 30 minutes.

Gelatinase test

To test for the presence of the gelatinase exoenzyme, a gelatinase test was performed. A gelatinase test agar plate was heavily inoculated in one quadrant using fresh broth culture. The plate was incubated at 37˚C for 24-48 hours, agar-side down. After this time, the plate was refrigerated at 1.6˚C for 10 minutes, and results were observed.

Lysine iron agar slant test

To test for the ability to decarboxylate or deaminate lysine and produce hydrogen sulfide, a lysine iron agar (LIA) slant test was performed. A fresh broth culture was used to stab the agar butt to the bottom twice and streak the slant. The tube with a loosened cap was incubated at 37˚C for 24-48 hours.

Coagulase test

To test for the presence of the coagulase enzyme, a coagulase test was performed using a loopful of fresh broth culture of the unknown organism. A colony paste was made using sterile water on top of a microscope slide, and a coagulase disk with rabbit serum was added, and the test was gently mixed. Sterile water was added, and the test mixed again. Results were observed within 30 seconds.

Oxidation/fermentation of dextrose test

To determine how the unknown organism metabolizes carbohydrate, an oxidation-fermentation test was performed using two gelatin oxidation-fermentation (O/F) agar tubes. A fresh broth culture was used to stab halfway to the bottom of both tubes. One tube had sterile mineral oil added to the surface of the media in a 1 cm layer and was incubated at 37˚C for 24-48 hours. The other tube was incubated at 37˚C for 24-48 hours with a loosened cap.

Results

Unknown sample number 30 showed a negative Gram staining reaction

After performing a Gram staining reaction, unknown sample 30 was found to be Gram-negative bacilli. The sample showed a distinctive rod-shape. (Figure 1)

A positive catalase test indicated the presence of the catalase enzyme in unknown 30

A positive catalase test was confirmed by the immediate formation of bubbles. (Data not shown)

A negative blood agar test indicated the absence of hemolytic exotoxins in unknown 30

A negative blood agar test was confirmed by the absence of any clearing of the agar around the colonies. The sample showed a distinctive gamma hemolysis. (Data not shown)

A positive spirit blue agar test indicted the presence of the lipase enzyme in unknown 30

A positive spirit blue agar test was confirmed by the clearing of the agar around the colonies. The sample showed a distinctive light area or halo that indicates the breakdown of the spirit blue/oil complex. (Data not shown)

A TSA plate at a different temperature indicated growth at a lower temperature and red pigmentation

A positive TSA plate at 25˚C test was confirmed by the presence of colony growth with concentrated red pigmentation. (Figure 2)

A negative SIM test indicated the absence of sulfide production, indole, and motility in unknown 30

A negative sulfide production SIM test was confirmed by the absence of any black precipitation. A negative indole test was confirmed by the yellow color of the added Kovac’s reagent. A negative motility test was confirmed by the lack of cloudiness or turbidity of the media. (Data not shown)

Unknown 30 appeared to be a facultative anaerobe

Twenty-four to forty-eight hours after incubation at 37˚C, the results of the fluid thioglycollate test were difficult to interpret. There was significant cloudiness at the top of the media, indicating that unknown 30 appeared to be a microaerophile. However, there was also some cloudiness at the middle and bottom of the media, indicating that unknown 30 appeared to be a facultative anaerobe. Given that there was turbidity in the middle and bottom of the media, which is absent in fluid thioglycollate tests positive for microaerophiles, a decision was made to determine the result as facultative anaerobe positive (+). (Data not shown)

A negative acid-fast stain indicated the absence of mycolic acid in the cell wall of unknown 30

A negative acid-fast staining reaction was confirmed by the presence of blue-stained cells. (Data not shown)

A positive Simmons-citrate test indicated the presence of the citrase enzyme in unknown 30

A positive Simmons-citrate test was confirmed by the change in media color from green to blue. (Data not shown)

Unknown 30 appeared to be lactose (+)

A positive triple sugar iron test was confirmed by the change in media color from red to yellow, indicating that unknown 30 can ferment any of the three sugars in the medium, lactose, sucrose, or glucose. A positive phenol red with lactose (fermentation) test was confirmed by the change in media color from red to yellow, indicating that unknown 30 can ferment lactose. (Data not shown)

Unknown 30 appeared to be sucrose (+)

A positive triple sugar iron test was confirmed by the change in media color from red to yellow, indicating that unknown 30 can ferment any of the three sugars in the medium, lactose, sucrose, or glucose. (Data not shown)

Unknown 30 appeared to be glucose (+)

A positive triple sugar iron test was confirmed by the change in media color from red to yellow, indicating that unknown 30 can ferment any of the three sugars in the medium, lactose, sucrose, or glucose. (Data not shown)

Unknown 30 appeared to be positive for gas production

A positive triple sugar iron test was confirmed by the presence of gas bubbles in the agar beneath the slant and throughout the medium. A positive phenol red with dextrose test was confirmed by the presence of gas in the Durham tube. A positive phenol red with lactose (fermentation) test was confirmed by the presence of gas in the Durham tube. (Data not shown)

Unknown 30 appeared to be dextrose (+)

A positive phenol red with dextrose test was confirmed by the change in media color from red to yellow. A positive oxidation / fermentation of dextrose test was confirmed by the change in media color from green to yellow, indicating that unknown 30 is capable of fermenting dextrose. (Data not shown)

A negative starch agar test indicated the absence of a-amylase and oligo-1,6-glucosidase in unknown 30

A negative starch agar test was confirmed by the absence of any clearing of the media around the colonies. (Data not shown)

A negative skim milk agar test indicated the absence of the casease exoenzyme in unknown 30

A negative skim milk agar test was confirmed by the absence of any clearing of the media around the colonies. (Data not shown)

A positive DNAse agar test indicated the presence of the deoxyribonuclease exoenzyme in unknown 30

A positive DNAse agar test was confirmed by the presence of a halo of cleared media around the colonies. (Data not shown)

A negative oxidase test indicated the absence of cytochrome c oxidases in unknown 30

A negative oxidase test was confirmed by the oxidase test swab remaining pink after being rolled over unknown 30 colonies. (Data not shown)

Unknown 30 appeared to grow in anaerobic conditions

A positive TSA incubation in an anaerobic jar was confirmed by the presence of colony growth. (Data not shown)

A negative nitrate reduction test indicated the absence of the nitrate reductase and nitrite reductase enzymes in unknown 30

A negative nitrite reductase test was confirmed by the absence of gas in the Durham tube. A ngative nitrate reductase test was confirmed by the absence of a media color change to red after the dropperful of sulfanilic and a-napthylamine were added. (Data not shown)

A negative phenylalanine deaminase test indicated the absence of the deaminase enzyme in unknown 30

A negative phenylalanine deaminase test was confirmed by the absence of a media color change from a dark yellow, cold color to green after the 10% ferric chloride was added. (Data not shown)

A negative methyl red-Voges Proskauer indicated the absence of the mixed acid fermentation pathway, butylene glycol pathway, and acetoin production in unknown 30

A negative methyl red test was confirmed by the color change of the methyl red to orange. A negative Voges-Proskauer test was confirmed by the lack of a color change to red after the a-napthol solution and potassium hydroxide solution was added. (Data not shown)

A negative urease test indicated the absence of the urease enzyme in unknown 30

A negative urease test was confirmed by the absence of a color change in which the media remained a peach color. (Data not shown)

A positive gelatinase test indicated the presence of the gelatinase enzyme in unknown 30

A positive gelatinase test was confirmed by the presence of liquid media after the plate had been refrigerated for ten minutes. (Data not shown)

Unknown sample number 30 showed a positive capsule staining reaction

After performing a capsule staining reaction, unknown sample 30 was found to have a capsule. (Figure 3)

Unknown 30 appeared to be lysine decarboxylation (+) and hydrogen sulfide production (+)

A positive lysine agar slant test for lysine decarboxylation was confirmed by the presence of a purple color throughout the media. A positive lysine agar test for hydrogen sulfide production was confirmed by the presence of black, wispy precipitate throughout the media. (Figure 4)

Unknown 30 appeared to be esculin hydrolyzation (+)

A positive bile esculin test was confirmed by the presence of darkening of the media by insoluble iron salts. (Data not shown)

A negative coagulase test indicated the absence of the coagulase enzyme in unknown 30

A negative coagulase test was confirmed by the absence of clumping within thirty seconds. (Data not shown)

Unknown 30 appeared to be fermentation (+)

A positive oxidation/fermentation of dextrose test was confirmed by a media color change to yellow in both tubes. A positive phenol red with lactose (fermentation) test was confirmed by the presence of a media color change to yellow. (Data not shown)

Figures and Tables

Table 1. All conducted test results of unknown 30, with correct literature results for tests pertinent for identification of unknown sample 30.

Test Results Literature Results

Gram stain Gram-negative, rod-shaped In agreement*

Catalase test Positive for catalase enzyme In agreement*

Blood agar Gamma hemolysis, negative for hemolytic exotoxins

Spirit Blue agar Positive for lipase enzyme

TSA plate at different temperature Positive for growth

SIM Negative for sulfide production In agreement*

Negative for indole In agreement*

Negative for motility (non-motile) Positive for motility (motile)*

Fluid Thioglycollate medium Positive for facultative anaerobe category

Acid Fast stain

Simmons-citrate test Positive for citrase enzyme In agreement*

Triple Sugar Iron test Positive for sugar fermentation (can ferment lactose, sucrose, glucose), positive for gas production Negative for lactose fermentation*

Phenol red with dextrose test Positive for dextrose utilization, positive for gas production

Starch agar Negative for α-amylase and oligo-1,6-glucosidase

Skim milk agar Negative for casease exoenzyme

DNAse agar Positive for deoxyribonuclease exoenzyme In agreement*

Oxidase test Negative for cytochrome c oxidases In agreement*

TSA incubation in anaerobic jar test Positive for growth in anaerobic conditions

Nitrate reduction test Negative for nitrate reductase Positive for nitrate reductase*

Negative for nitrite reductase Positive for nitrite reductase

Phenylalanine deaminase test Negative for deaminase enzyme In agreement*

MR-VP (methyl red Voges-Proskauer) test Negative for mixed acid fermentation pathway In agreement*

Negative for butylene glycol pathway and acetoin production Positive for butylene glycol pathway and acetoin production*

Urease test Negative for urease enzyme In agreement*

Gelatinase test Positive for gelatinase enzyme In agreement*

Capsule staining Positive for capsule

Lysine Iron agar slant Positive for lysine decarboxylation In agreement*

Positive for hydrogen sulfide production In agreement

Bile esculin test Positive for esculin hydrolyzation In agreement*

Coagulase test Negative for coagulase enzyme

Oxidation / fermentation of dextrose test Positive for fermentation of dextrose, fermentative In agreement*

Phenol red with lactose (fermentation) test Positive for lactose fermentation and gas production Negative for lactose fermentation*

*(Holt et al. 1994)

Figure 1. A negative Gram-staining of unknown 30 confirmed the presence of uniformly rod-shaped bacteria.

Figure 2. A positive TSA plate at 25˚C was confirmed by red-pigmented colony growth.

Figure 3. A positive capsule stain was confirmed by the presence of a white halo around the cells of unknown 30.

Figure 4. A positive lysine iron agar slant test indicated unknown 30 was lysine decarboxylation (+), which was confirmed by the purple color of the media.

Discussion

Identification

Unknown 30 was identified as Serratia marcescens (Figure 5, Figure 6). However, due to a contamination issue in the lab, the identification process was conducted using literature results. Only tests pertinent to the identification of Serratia marcescens will be expounded upon in this section. For all tests results, please refer to Table 1.

To identify unknown 30, the first test performed was a Gram-stain. The negative, uniformly bacillary shape of the results indicated that unknown 30 could belong to the family Enterobacteriaceae, Pseudomonas, Aeromonas, Alcaligens, or Chromobacterium (Holt et al. 1994). An oxidase test with negative results confirmed that unknown 30 belonged to the Enterobacteriaceae family (Holt et al. 1994). Next, the negative results of the lactose fermentation tests triple sugar iron test and phenol red with lactose test indicated that unknown 30 could be Edwardsiella tarda, Erwinia cacticida, Morganella morganii, Proteus mirabilis, Proteus penneri, Proteus vulgaris, Providencia stuartii, Salmonella bongori, Salmonella enterica, Serratia marcescens, Serratia liquefaciens, Shigella spp. (boydii, dysenteriae, flexneri), Shigella sonnei, Yersinia enterocolitica, Yersinia pestis, or Yersinia pseudotuberculosis (Holt et al. 1994). A negative indole of the SIM test narrowed the possibilities to Erwinia cacticida, Proteus mirabilis, Proteus penneri, Salmonella bongori, Salmonella enterica, Serratia marcescens, Serratia liquefaciens, Shigella sonnei, Yersinia pestis, or Yersinia pseudotuberculosis (Holt et al. 1994). A negative urease test further narrowed the identity of unknown 30 to Erwinia cacticida, Salmonella bongori, Salmonella enterica, Serratia marcescens, Serratia liquefaciens, Shigella sonnei, or Yersinia pestis (Holt et al. 1994). A positive motility of the SIM test limited the identity to Erwinia cacticida, Salmonella bongori, Salmonella choleraesuis, Serratia marcescens, or Serratia liquefaciens, and a positive lysine decarboxylation test of the lysine iron agar slant only eliminated Erwinia cacticida (Holt et al. 1994). The negative hydrogen sulfide production test rendered the identity of unknown 30 as either Serratia marcescens, or Serratia liquefaciens (Holt et al. 1994). The TSA plate incubated at a different temperature indicated that unknown 30 had a red pigmentation, identifying unknown 30 as Serratia marcescens (Holt et al. 1994).

Figure 5. Tests used in the identification of Serratia marcescens. (Holt et al. 1994).

Figure 6. An alternative testing route in the identification of Serratia marcescens.

Serratia marcescens description

Serratia marcescens are Gram-negative straight rods that are approximately 0.5-0.8 mm in diameter and 0.9-2.0 mm in length (Holt et al. 1994). They are facultatively anaerobic, chemoorganotrophic, and have both respiratory and fermentative types of metabolism (Holt et al. 1994). They are also made motile by peritrichous flagella (Holt et al. 1994)

Habitat

S. marcescens has a widely distributed habitat, and has been found in starch-rich foods (Hejazi and Falkiner 1997). In addition to food, this microorganism has also been found in a variety of ecological niches, such as soil, water, air, plants, animals, and humans (Hejazi and Falkiner 1997). It is also able to survive and grow in extreme conditions, such as in areas despite the presence of disinfectants, antiseptics, and doubly distilled water (Hejazi and Falkiner 1997). S. marcescens also grows well in damp environments, and can be found in bathrooms, particularly on substances such as tile grout, shower corners, toilet water line, and basins (Wilfert et al. 1970).

Pathogenesis

S. marcescens is a contributor to hospital-acquired infections and can cause respiratory tract infections, urinary tract infections, septicemia, meningitis, and wound infections (Hejazi and Falkiner 1997). It has also been found to cause infective endocarditis and usually affects the left side of the heart, in contrast with other Gram-negative bacteria (Hejazi and Falkiner 1997).

Most strains of S. marcescens are resistant to multiple antibiotics due to the presence of R-factors, a type of plasmid that carry genes that encode for resistance (Wilfert et al. 1970)

Conclusion

The identification of bacteria is vital to many disciplines, especially to medical professionals in the healthcare field. By conducting a series of characteristic tests, particularly a Gram-stain, oxidase test, triple sugar iron test, phenol red with lactose test, SIM test, urease test, lysine iron agar slant test, and a TSA plate at 25˚C test, unknown sample 30 was identified as Serratia marcescens. This bacteria is a human pathogen that is capable of causing a variety of infections, such as respiratory tract infections, urinary tract infection, and wound infections, among others.