MICRO BIO 125 – Identifying bacteria can lead to many new discoveries such as new species

15 Jun MICRO BIO 125 – Identifying bacteria can lead to many new discoveries such as new species

Question
INTRODUCTION

The identification of an unknown is beneficial in many ways, including the identification of bacteria in diseases, and in research. Identifying bacteria can lead to many new discoveries such as new species or the evolution of a known species. In this experiment, an unknown bacterium was to be identified using a number of different tests and using several different bacteria for comparison. The unknown was tested with gram staining, capsule staining, motility test, oxygen/fermentation tests, hydrolytic and degradative tests. The unknown was also tested for different morphological and cultural characteristics. The outcome of these test indicated that the assigned unknown is Enterobacter aerogenes.

MATERIALS AND METHODS

This experiment was conducted over a period of three weeks, six days in total. On day one an unknown in a tryptic soy agar slant labeled as number two was randomly assigned. A macroscopic purity check was conducted first to note any unique cultural characteristics. Table 1 lists further tests, reagents, purpose, and results used in the identification processes. Note all of these tests and procedures were performed as stated in the course laboratory manual by MSJC (1), unless otherwise noted.

Table 1: Biochemical Tests performed

Date Inoculated

Test Performed

Purpose or Objective of the Test

Materials & Methods Used

Temperature and Time of Incubation

Positive Control

Negative Control

04/19/16

Tuesday

(2) Tryptic Soy Agar Slants

To grow bacterium for stock and acquire its physical characteristics (growth pattern)

· Numbered Unknown “Original” bacteria culture

· 2 Tryptic Soy Agar slant tubes (TSA)

· Inoculating Loop

· Incinerator

Temperatures: 25°C and 37°C

Time of Incubation: 18-24 hours

None

None

(3) Tryptic Soy Broths

To acquire bacterium’s physical characteristics (growth pattern & optimum growth temperature)

· Numbered Unknown “Original” bacteria culture

· 3 Tryptic Soy Broths (TSB)

· Inoculating Loop

· Incinerator

Temperatures:

25°C, 30°C, 37°C

Time of Incubation: 18-24 hours

None

None

Blood Agar Plate

To acquire bacterium’s physical characteristics (colony morphology & hemolysis type)

· Numbered Unknown “Original” bacteria culture

· 1 Blood Agar Plate

· Inoculating Loop

· Incinerator

Temperature:

37°C

Time of Incubation: 24 hours

**No Postitive or Negative Controls for Blood Agar Plate

Beta Hemolysis Control: Staphylococcus aureus

Gamma Hemolysis Control:

Acinetobacter baumannii

Tryptic Soy Agar Plate

To acquire bacterium’s physical characteristics (colony morphology)

· Numbered Unknown “Original” bacteria culture

· 1 Tryptic Soy Agar Plate

· Inoculating Loop

· Incinerator

Temperature:

37°C

Time of Incubation: 24 hours

None

None

Gram Stain – Purity Check

To determine if the original stock of the bacterium is a pure culture

· Numbered Unknown “Original” bacteria culture

· Clean microscope slides

· Staining Kit

· Inoculating Loop

· Incinerator

· Microscope Slide Rack

· Marking pen

None

None

None

4/21/16

Thursday

Thioglycollate w/Indicator (FTM) Broth

To determine oxygen requirements of the unknown bacterium

· Working stock of Unknown bacterium

· 1 Thioglycollate w/Indicator (FTM) tube

· Inoculating Needle

· Incinerator

· Marker

· Test Tube Rack

Temperature:

37°C

Time of Incubation: 120 hours (5 days)

None

None

Gelatinase Production (Liquefication)

To determine if the bacterium possesses the ability to hydrolyze gelatin

· Working stock of Unknown bacterium

· 1 Gelatin Agar Deep tube

· Inoculating Needle

· Incinerator

· Marker

· Test Tube Rack

Temperature:

37°C

Time of Incubation: 264 hours (11 days)

Bacillus subtilis

Escherichia coli

Motility Agar Deep

To determine if the bacterium is motile or nonmotile

· Working stock of Unknown bacterium

· 1 Motility Agar Deep tube

· Inoculating Needle

· Incinerator

· Marker

· Test Tube Rack

Temperature:

37°C

Time of Incubation: 24 hours

Escherichia coli

Klebsiella pneumoniae

Urease Production (Hydrolysis)

To determine whether or not the bacterium produces urea as a product of amino acid metabolism

· Working stock of Unknown bacterium

· 1 Urea Agar Slant

· Inoculating Loop

· Incinerator

· Marker

· Test Tube Rack

Temperature:

37°C

Time of Incubation: 264 hours (11 days)

Proteus vulgaris

Escherichia coli

Gram Stain w/Control

To determine and compare the morphology, cell arrangement and size of the unknown bacterium to S. aureus

· Working stock of Unknown bacterium

· Tryptic Soy Broth Culture of S. aureus

· Clean microscope slides

· Staining Kit

· Inoculating Loop

· Incinerator

· Deionized water

· Microscope Slide Rack

· China Marker

None

None

None

Capsule Stain

To determine if the bacterium possesses a capsule

· Working stock of Unknown bacterium

· Clean microscope slides

· India Ink & Crystal Violet dyes

· Inoculating Loop

· Incinerator

· Deionized water

· Microscope Slide Rack

· Microscope

None

None

None

Motility Wet Mount

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Catalase test

Oxidase test

To determine what kind of motility the bacterium exhibits

To determine oxygen requirements

To determine oxygen requirements

· Working stock of Unknown bacterium

· Clean microscope slides and cover slips

· Inoculating Loop

· Incinerator

· Deionized water

· Microscope

· Clean microscope slide

· Wooden applicator stick

· TSA plate culture of unknown

· 1 drop of Catalase reagent (H2O2)

· Clean watch glass

· Wooden applicator stick

· 1 piece Watman Filter Paper

· 1 drop per Oxidase Reagent

· TSA plate culture of Unknown

None

None

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None

None

Staphylococcus aureus

Pseudomonas aeruginosa

None

None

None

04/26/16

Tuesday

Nitrate Reduction

To determine if the bacterium can reduce nitrate to nitrite or nitrogen gas

· Working stock of Unknown bacterium

· 1 Nitrate Broth with Durham tube

· Inoculating Loop

· Incinerator

· Nitrate Reagents A and B (5 drops of each)

· Marking pen

Temperature:

37°C

Time of Incubation: 72 hours (3 days)

Escherichia coli

Acinetobacter baumanii

Simmon’s Citrate Test

To determine if the bacterium utilizes citrate to produce ammonia as a by-product

· Working stock of Unknown bacterium

· Simmon’s Citrate Agar Slant

· Inoculating Loop

· Marking pen

Temperature:

37°C

Time of Incubation: 48 hours (2 days)

Enterobacter aerogenes

Escherichia coli

Indole Test (Tryptophan Degradation)

To determine if the bacterium can break down tryptophan and produce indole as a product

· Working stock of Unknown bacterium

· 1 Tryptone Broth Tube

· Inoculating Loop

· Incinerator

· Test Tube Rack

· 5 Drops of Kovac’s Reagent

· Marking pen

Temperature:

37°C

Time of Incubation: 48 hours (2 days)

Escherichia coli

Enterobacter aerogenes

Methyl Red Test

To determine if there is a high concentration of hydrogen ions (acid) after the fermentation of glucose

· 1 milliliter of MVRP broth tube broth

· Inoculating Loop

· Incinerator

· Clean Test Tube

· 3-4 drops of Methyl Red Reagent

· Marking pen

Temperature:

37°C

Time of Incubation: 48 hours (2 days)

Escherichia coli

Enterobacter aerogenes

Voges-Proskauer Test

To determine whether or not acetoin is present in the broth solution

· 1 milliliter of MVRP broth tube broth

· Inoculating Loop

· Incinerator

· Clean Test Tube

· VP Reagents A (15 drops) & B (5 drops)

· Marking pen

Temperature:

37°C

Time of Incubation: 48 hours (2 days)

Enterobacter aerogenes

Escherichia coli

04/28/16

Thursday

Carbohydrate Fermentation Tests

To determine the presence of acid and gas in the inoculated cultures from the fermentation of specific types of sugars (indicated by a color change)

· Working stock of Unknown bacterium

· Inoculating Loop

· Incinerator

· 1 Phenol Red Glucose broth with Durham tube

· 1 Phenol Red Lactose broth with Durham tube

· 1 Phenol Red Mannitol broth with Durham tube

· 1 Phenol Red Sucrose broth with Durham tube

· Marking pen

· Test Tube Rack

Temperature:

37°C

Time of Incubation: 24 hours

Escherichia coli

Alcaligenesfaecalis

Oxidative/Fermentative Metabolism Tests

To determine whether or not the unknown bacterium has oxidative and/or fermentative metabolism

· Working stock of Unknown bacterium

· 2 O/F Glucose Tubes

· Inoculating Loop

· Sterile Mineral Oil for overlay

· Incinerator

· Marking pen

Temperature:

37°C

Time of Incubation: 48 hours (2 days)

Escherichia coli (O and F)

Pseudomonas aeruginosa(O not F)

H₂S Production Test

To determine if the unknown bacterium produces hydrogen sulfide in the presence of glucose and lactose

· Working stock of Unknown bacterium

· Kligler’s Iron Agar Slant

· Incinerator

· Inoculating Needle (used to both stab and streak the slant)

Temperature:

37°C

Time of Incubation: 48 hours (2 days)

Proteus vulgaris

Escherichia coli

05/02/16

Tuesday

Amino Acid Decarboxylation

To determine if the bacterium can produce the enzyme decarboxylase in the presence of different amino acids (ornithine, arginine, and lysine)

· Working stock of Unknown bacterium

· 1 Moeller’s Ornithine Decarboxylase Broth Tube

· 1 Moeller’s Arginine Decarboxylase Broth Tube

· 1 Moeller’s Lysine Decarboxylase Broth Tube

· Inoculating Loop

· Incinerator

· Sterile Mineral Oil for overlay

· Marking pen

Temperature:

37°C

Time of Incubation: 48 hours

Escherichia coli

Proteus vulgaris

Starch Hydrolysis (Amylase Production)

To determine if the bacterium produces sugar in the presence of starch

· Working stock of Unknown bacterium

· 1 Starch Agar Plate

· Inoculating Loop

· Incinerator

· Iodine solution (as a starch indicator)

· Marking pen

Temperature:

37°C

Time of Incubation: 48 hours

Bacillus subtilis

Escherichia coli

Casein Hydrolysis (Protease Production)

To determine the presence of caseinase in the presence of milk protein

· Working stock of Unknown bacterium

· 1 Skim Milk Agar Plate

· Inoculating Loop

· Incinerator

· Marking pen

Temperature:

37°C

Time of Incubation: 48 hours

Bacillus cereus

Escherichia coli

DNase Production

To determine whether or not the bacterium is capable of producing DNase

· Working stock of Unknown bacterium

· 1 DNase Agar Plate

· Inoculating Loop

· Incinerator

· Dropper Bottle if 5M Hydrochloric Acid (HCI) (used to flood the plate)

Temperature:

37°C

Time of Incubation: 24 hours

Staphylococcus aureus

Alcaligenesfaecalis

Phenylalanine Deamination (Phenylalanase Production)

To determine whether or not the bacterium has produced the enzyme phenylalanine deaminase

· Working stock of Unknown bacterium

· 1 Phenylalanine Agar Slant

· Inoculating Loop

· Incinerator

· Dropping Bottle of 10% Ferric Chloride (3-5 drops)

Temperature:

37°C

Time of Incubation: 24 hours

Proteus vulgaris

Enterobacter aerogenes

RESULTS

Unknown two had morphology on a tryptic soy agar plate as small sized opaque white colored colony, and γ hemolytic as determined from the blood agar plate. After determining the unknown to be a Gram negative rod figure 18.1 in appendix F was eliminated.

Table 2: Outcomes of Biochemical Tests and Comparison of K. pneumoniae&A. baumanii

(Cultural) Test Performed

Observations

Interpretation of Results

Enterobacter aerogenes

Enterobacter cloacae

Oxidase Test

No change

–

–

–

Catalase Test

Bubbles upon contact

+

+

+

Thioglycollate w/Indicator

Turbidity in the top half of broth

Facultative Anaerobe

Facultative Anaerobe

Facultative Anaerobe

Gelatinase Production

Did not liquefy

–

–

d

Motility Agar Deep

Motile

+

+

+

Urease Production

Salmon color

–

–

v

Capsule Stain

Capsule observed

+

+

+

Nitrate Reduction

Red color after adding of Reagents A & B (no zinc)

+

+

–

Simmon’s Citrate

Blue Agar after Incubation

+

+

+

Indole

None

–

–

–

MR Test

Yellow color change

–

–

+

VP Test

Pink color change

+

+

–

Carbohydrate Fermentation (Glucose, Lactose, Mannitol, Sucrose)

Yellow/Bubbles for Glucose

AG

AG

+

Yellow/Bubble for Lactose

AG

AG

+

Yellow/Bubbles for Mannitol

AG

AG

–

Yellow/Bubbles for Sucrose

AG

AG

–

O/F Metabolism

Oxidative & Fermentative

+/+

+/+

+/-

H₂S Production

Red over Yellow Agar

–

–

–

Amino Acid Decarboxylation (Lysine, Ornithine, Arginine)

Purple color for Lys.

+

+

–

Purple color for Orn.

+

+

+

Yellow color for Arg.

–

–

+

Amylase Production

No change observed

–

–

[d]

Casein Hydrolysis

No change observed

–

–

–

DNase Production

No change observed

–

–

–

Phenylalanase Production

Yellow color observed

–

–

+

DISCUSSION