MIXED UNKNOWNS

LEARNING OBJECTIVES

Demonstrate competency of microbiology laboratory techniques by separating and identifying two organisms in a mixed broth

Design two dichotomous keys to differentiate the possible Gram-negative unknowns and the possible Gram-positive unknowns

MCCCD OFFICIAL COURSE COMPETENCIES

Describe the modes of bacterial and viral reproduction and proliferation

Utilize aseptic technique for safe handling of microorganisms

Apply various laboratory techniques to identify types of microorganisms

Identify structural characteristics of the major groups of microorganisms

Compare and contrast prokaryotic cell and eukaryotic cell

Compare and contrast the physiology and biochemistry of the various groups of microorganisms

MATERIALS

Unknown mixed broth containing Gram-positive cocci and Gram-negative bacilli

1 MacConkey Agar

1 CNA Agar

Inoculating loop

Sterile swab

BIOCHEMICAL TESTS ALBUM LINK

Possible Gram-negative unknowns:        Possible Gram-positive unknowns    

Pseudomonas aeruginosa                                    Staphylococcus aureus

Alcaligenes faecalis                                                 Staphylococcus epidermidis

Escherichia coli                                                         Micrococcus luteus

Klebsiella aerogenes                                               Micrococcus roseus

Proteus mirabilis                                                      Group C Streptococcus

Klebsiella pneumoniae                                          Streptococcus pyogenes

Salmonella enterica                                                Streptococcus pneumoniae

Shigella flexneri                                                        Streptococcus sanguinis

                                                                                         Enterococcus faecalis

 

There are two different types of bacteria in the broth of the mixed unknown. One type is Gram-positive cocci and the other is Gram-negative bacilli. First, you must isolate and separate the two organisms from each other by using selective media.

The MacConkey Agar is selective for Gram-negative organisms. The crystal violet dye and the bile salts in the media inhibit the growth of Gram-positive organisms. MacConkey agar is also a differential media because it contains lactose. Gram-negative bacilli that ferment lactose will appear hot pink and those that do not ferment lactose will appear colorless.

The Colistin-Naladixic Agar is selective for Gram-positive organisms. CNA contains the antibiotics colistin and naladixic acid which suppress the growth of Gram-negative organisms. CNA is differential because it contains red blood cells. You can determine the hemolytic properties of the Gram-positive cocci.

Once the bacteria have been isolated from each other, the next step is to identify both bacteria. By using biochemical test charts to differentiate the Gram-positive cocci and the Gram-negative bacilli, you will design two dichotomous keys (flow charts) to guide you in determining which tests will help you identify your unknowns. The result of the first test will determine the next test to perform.

You must complete the tests in the order of the dichotomous key until you reach an identification of your unknowns. Record the biochemical tests you perform in the order of your dichotomous key. Record the identification of the unknown using correct binomial nomenclature.

PRE-ASSESSMENT

PROCEDURE

Day 1: streak plates of selective/differential media

traditional method streak plate

1.  Label the MacConkey and CNA plates with your name (not initials), date, unknown number and the name of the media. Record your unknown number on the worksheet.

2.  Divide the MacConkey and CNA plates into 3 sections by drawing a “T” on the bottom of the plate where the media is.

Divide the plate into 3 sections for the streak plate3.  Resuspend the mixed unknown broth culture by gently shaking or tapping the tube side to side until the bacteria swirl up from the bottom of the tube.

MacConkey Agar Streak Plate

1. Peel the paper cover from the wooden end of a sterile swab. Handle ONLY the wooden end of the swab to maintain the sterility of the cotton end.

2.  Holding the tube in your non-dominant hand, remove the cap of the bacterial culture with your little finger on your dominant hand. Dip the cotton end of the swab into the mixed unknown broth and then slightly press the swab against the inside of the tube to expel any excess broth. Recap the bacterial culture tube and place it in a test tube rack.

3.  Using your non-dominant hand, open the lid of the plate just enough to fit the swab in.

Lifting lid of Petri plate to insert a swab

4.  Inoculate the first section of the plate by touching the swab down on the surface of the agar and drawing a zig-zag streak to fill that first area of the plate. Replace the Petri plate lid. Dispose of the swab in the autoclave trash.

Traditional streak plate section 1

5.  Use an inoculating loop to complete the rest of the procedure.

6.  Sterilize your inoculating loop in the Bacticinerator and allow it to cool.

7.  Open the lid of the plate just enough to fit the loop in. Place the loop in the center of the first section and gently drag the loop one time into the second section. Lightly drag the tip of the loop from side to side in a back-and-forth motion to spread the inoculum to fill the second section. Replace the Petri plate lid.

Traditional streak plate section 28.  Sterilize the loop in the Bacticinerator and allow it to cool.

9.  Open the lid of the plate just enough to fit the loop in. Place the loop in the center of the second section and gently drag the loop one time into the third section. Lightly drag the tip of the loop from side to side in a back-and-forth motion to spread the inoculum to fill the third section. Replace the Petri Plate lid.

Traditional streak plate section 3

10.  Sterilize the loop in the Bacticinerator and allow it to cool.

11.  Incubate your MacConkey agar plate lid side down in the class plate tray until the next lab period.

CNA Agar Streak Plate

1. Peel the paper cover from the wooden end of a sterile swab. Handle ONLY the wooden end of the swab to maintain the sterility of the cotton end.

2.  Holding the tube in your non-dominant hand, remove the cap of the bacterial culture with your little finger on your dominant hand. Dip the cotton end of the swab into the mixed unknown broth and then slightly press the swab against the inside of the tube to expel any excess broth. Recap the bacterial culture tube and place it in a test tube rack.

3.  Using your non-dominant hand, open the lid of the plate just enough to fit the swab in.

Lifting lid of Petri plate to insert a swab

4.  Inoculate the first section of the plate by touching the swab down on the surface of the agar and drawing a zig-zag streak to fill that first area of the plate. Replace the Petri plate lid. Dispose of the swab in the autoclave trash.

Traditional streak plate section 1

5.  Use an inoculating loop to complete the rest of the procedure.

6.  Sterilize your inoculating loop in the Bacticinerator and allow it to cool.

7.  Open the lid of the plate just enough to fit the loop in. Place the loop in the center of the first section and gently drag the loop one time into the second section. Lightly drag the tip of the loop from side to side in a back-and-forth motion to spread the inoculum to fill the second section. Replace the Petri plate lid.

Traditional streak plate section 28.  Sterilize the loop in the Bacticinerator and allow it to cool.

9.  Open the lid of the plate just enough to fit the loop in. Place the loop in the center of the second section and gently drag the loop one time into the third section. Lightly drag the tip of the loop from side to side in a back-and-forth motion to spread the inoculum to fill the third section. Replace the Petri Plate lid.

Traditional streak plate section 3

10.  Sterilize the loop in the Bacticinerator and allow it to cool.

11.  Stab the sterilized loop 2–3 times into the agar in the first section of the streak plate. Stabbing into the media will enhance the appearance of hemolysis.

12.  Incubate your CNA plate lid side down in a candle jar since some of the unknown Gram-Positive organisms are microaerophilic.

13.  Label your mixed unknown broth with your name and lab section (day & time). Place the broth in the rack. These tubes will be saved in the refrigerator in case your unknowns do not grow on the CNA or MacConkey agar plates.

alternative method streak plate

1.  Label the MacConkey and CNA plates with your name (not initials), date, unknown number and the name of the media. Record your unknown number on the worksheet.

2.  Divide the MacConkey and CNA plates into 3 sections by drawing a “T” on the bottom of the plate where the media is.

Dividing Petri plate into 3 sections

3.  Resuspend the mixed unknown broth culture by gently shaking or tapping the tube side to side until the bacteria swirl up from the bottom of the tube.

MacConkey Agar Streak Plate

1. Peel the paper cover from the wooden end of a sterile swab. Handle ONLY the wooden end of the swab to maintain the sterility of the cotton end.

2.  Holding the tube in your non-dominant hand, remove the cap of the bacterial culture with your little finger on your dominant hand. Dip the cotton end of the swab into the mixed unknown broth and then slightly press the swab against the inside of the tube to expel any excess broth. Recap the bacterial culture tube and place it in a test tube rack.

3.  Using your non-dominant hand, open the lid of the plate just enough to fit the swab in.

Lifting lid of Petri plate to insert a swab

4.  Inoculate the first section of the plate by touching the swab down on the surface of the agar and drawing a zig-zag streak to fill that first area of the plate. Replace the Petri plate lid. Dispose of the swab in the autoclave trash.

Alternative streak plate section 1

5.  Use an inoculating loop to complete the rest of the procedure.

6.  Sterilize your inoculating loop in the Bacticinerator and allow it to cool.

7.  Open the lid of the plate just enough to fit the loop in. Inoculate the second section of the plate by touching the loop down in the middle of the first section of the plate and draw a line toward the second section.  Continue to draw parallel lines to fill the second section. Replace the Petri plate lid.

Alternative streak plate section 2

8.  Sterilize your inoculating loop in the Bacticinerator and allow it to cool.

9.  Open the lid of the plate just enough to fit the loop in. Inoculate the third section of the plate by touching the loop down by touching down in the middle of the second section of the plate and draw a line toward the third section.  Continue to draw parallel lines to fill the third section. Replace the Petri plate lid.

Alternative Streak Plate Section 3

10.  Sterilize your inoculating loop in the Bacticinerator and allow it to cool.

11.  Incubate your MacConkey agar plate lid side down in the class plate tray until the next lab period.

CNA Agar Streak Plate

1. Peel the paper cover from the wooden end of a sterile swab. Handle ONLY the wooden end of the swab to maintain the sterility of the cotton end.

2.  Holding the tube in your non-dominant hand, remove the cap of the bacterial culture with your little finger on your dominant hand. Dip the cotton end of the swab into the mixed unknown broth and then slightly press the swab against the inside of the tube to expel any excess broth. Recap the bacterial culture tube and place it in a test tube rack.

3.  Using your non-dominant hand, open the lid of the plate just enough to fit the swab in.

Lifting lid of Petri plate to insert a swab

4.  Inoculate the first section of the plate by touching the swab down on the surface of the agar and drawing a zig-zag streak to fill that first area of the plate. Replace the Petri plate lid. Dispose of the swab in the autoclave trash.

Alternative streak plate section 1

5.  Use an inoculating loop to complete the rest of the procedure.

6.  Sterilize your inoculating loop in the Bacticinerator and allow it to cool.

7.  Open the lid of the plate just enough to fit the loop in. Inoculate the second section of the plate by touching the loop down in the middle of the first section of the plate and draw a line toward the second section.  Continue to draw parallel lines to fill the second section. Replace the Petri plate lid.

Alternative streak plate section 2

8.  Sterilize your inoculating loop in the Bacticinerator and allow it to cool.

9.  Open the lid of the plate just enough to fit the loop in. Inoculate the third section of the plate by touching the loop down by touching down in the middle of the second section of the plate and draw a line toward the third section.  Continue to draw parallel lines to fill the third section. Replace the Petri plate lid.

Alternative Streak Plate Section 3

10.  Sterilize your inoculating loop in the Bacticinerator and allow it to cool.

11.  Stab the sterilized loop 2–3 times into the agar in the first section of the streak plate. Stabbing into the media will enhance the appearance of hemolysis.

12.  Incubate your CNA plate lid side down in a candle jar since some of the unknown Gram-Positive organisms are microaerophilic.

13.  Label your mixed unknown broth with your name and lab section (day & time). Place the broth in the rack. These tubes will be saved in the refrigerator in case your unknowns do not grow on the CNA or MacConkey agar plates.

Day 2:  Make Stock Cultures

gram-negative bacilli unknown

MACCONKEY AGAR AFTER INCUBATION – Observe your MacConkey Agar plate and determine if your organism ferments lactose and record your results on the mixed unknown worksheet. If you do not have growth, reincubate your plates and subculture the original broth again.

Hot pink colonies (look at the color of the colonies not the color of the media) = Positive for lactose fermentation

Clear colonies = Negative for lactose fermentation (some lactose negative colonies may have a slight pink color due to the transmittance of the media color through the colony)

Positive lactose fermentation on MacConkey agar
Positive lactose fermentation results on MacConkey agar. Note the pink colonies on all three plates.
Positive lactose fermentation on MacConkey agar
Negative lactose fermentation results on MacConkey agar. Note the clear colonies.

MAKE THE GRAM-NEGATIVE STOCK CULTURE traditional streak plate

1. Label a TSA plate with your name, your unknown number, and the words “Gram Negative Stock”. Divide the plate into 3 sections by drawing a “T” on the plate. THIS WILL BE YOUR GRAM NEGATIVE STOCK CULTURE. You will use this stock culture to inoculate all future biochemical tests performed on this unknown. TSA is a “non-inhibitory” media which allows the organism to produce enzymes and metabolize the ingredients in the media.

2.  Sterilize your inoculating loop in the Bacticinerator and allow it to cool thoroughly.

3.  Use the inoculating loop take ONE SINGLE ISOLATED colony from the MacConkey Agar.

4.  Open the lid of the TSA plate just enough to fit the loop in. Inoculate the first section of the plate by touching the loop down on the surface of the agar and drawing a zig-zag streak to fill that first area of the plate. Replace the Petri plate lid.

5.  Sterilize your inoculating loop in the Bacticinerator and allow it to cool.

6.  Open the lid of the plate just enough to fit the loop in. Place the loop in the center of the first section and gently drag the loop one time into the second section. Lightly drag the tip of the loop from side to side in a back-and-forth motion to spread the inoculum to fill the second section. Replace the Petri plate lid.

7.  Sterilize the loop in the Bacticinerator and allow it to cool.

8.  Open the lid of the plate just enough to fit the loop in. Place the loop in the center of the second section and gently drag the loop one time into the third section. Lightly drag the tip of the loop from side to side in a back-and-forth motion to spread the inoculum to fill the third section. Replace the Petri Plate lid.

9.  Sterilize the loop in the Bacticinerator and allow it to cool.

10.  Incubate the Gram-Negative TSA stock plate lid side down in the class plate tray until the next lab period.

11.  Place the MacConkey plate lid side down in the class tray to be saved in the refrigerator in case your Gram-Negative stock plate does not grow.

MAKE THE GRAM-NEGATIVE STOCK CULTURE Alternative streak plate

1. Label a TSA plate with your name, your unknown number, and the words “Gram Negative Stock”. Divide the plate into 3 sections by drawing a “T” on the plate. THIS WILL BE YOUR GRAM NEGATIVE STOCK CULTURE. You will use this stock culture to inoculate all future biochemical tests performed on this unknown. TSA is a “non-inhibitory” media which allows the organism to produce enzymes and metabolize the ingredients in the media.

2.  Sterilize your inoculating loop in the Bacticinerator and allow it to cool thoroughly.

3.  Use the inoculating loop take ONE SINGLE ISOLATED colony from the MacConkey Agar.

4.  Using your non-dominant hand, open the lid of the plate just enough to fit the loop in.

5.  Inoculate the first section of the plate by touching the loop down on the surface of the agar and drawing a zig-zag streak to fill that first area of the plate. Replace the Petri plate lid.

6.  Sterilize your inoculating loop in the Bacticinerator and allow it to cool.

7.  Open the lid of the plate just enough to fit the loop in. Inoculate the second section of the plate by touching the loop down in the middle of the first section of the plate and draw a line toward the second section.  Continue to draw parallel lines to fill the second section. Replace the Petri plate lid.

8.  Sterilize your inoculating loop in the Bacticinerator and allow it to cool.

9.  Open the lid of the plate just enough to fit the loop in. Inoculate the third section of the plate by touching the loop down by touching down in the middle of the second section of the plate and draw a line toward the third section.  Continue to draw parallel lines to fill the third section. Replace the Petri plate lid.

10.  Sterilize your inoculating loop in the Bacticinerator and allow it to cool.

11.  Incubate the Gram-Negative TSA stock plate lid side down in the class plate tray until the next lab period.

12.  Place the MacConkey plate lid side down in the class tray to be saved in the refrigerator in case your Gram-Negative stock plate does not grow.

GRAM-POSITIVE COCCI UNKNOWN

AFTER INCUBATION – Evaluate the type of hemolysis produced by the organisms.  Hold the plate so that light is shining through the back of the agar. Determine the extent of damage to the blood in the agar surrounding the bacterial colonies. If condensation is preventing you from seeing the hemolysis, lift the lid of the plate just enough to insert a sterile swab. Use the sterile swab to remove the condensation. Record the hemolysis results on the mixed unknown worksheet.

Beta hemolysis = complete clearing of the red blood cells in the media
Beta hemolysis complete clearing of the red blood cells in the media
Alpha hemolysis = olive green discoloration of the media
Alpha hemolysis olive green discoloration of the media
Gamma hemolysis = no damage to the media
Gamma hemolysis = no damage to the media

MAKE THE GRAM-positive STOCK CULTURE traditional streak plate

1. Label a TSA plate with your name, your unknown number, and the words “Gram Positive Stock”. Divide the plate into 3 sections by drawing a “T” on the plate. THIS WILL BE YOUR GRAM POSITIVE STOCK CULTURE. You will use this stock culture to inoculate all future biochemical tests performed on this unknown. TSA is a “non-inhibitory” media which allows the organism to produce enzymes and metabolize the ingredients in the media.

2.  Sterilize your inoculating loop in the Bacticinerator and allow it to cool thoroughly. Use the inoculating loop take ONE SINGLE ISOLATED colony from the CNA Agar.

3.  Open the lid of the TSA plate just enough to fit the loop in. Inoculate the first section of the plate by touching the loop down on the surface of the agar and drawing a zig-zag streak to fill that first area of the plate. Replace the Petri plate lid.

4.  Sterilize your inoculating loop in the Bacticinerator and allow it to cool.

5.  Open the lid of the plate just enough to fit the loop in. Place the loop in the center of the first section and gently drag the loop one time into the second section. Lightly drag the tip of the loop from side to side in a back-and-forth motion to spread the inoculum to fill the second section. Replace the Petri plate lid.

6.  Sterilize the loop in the Bacticinerator and allow it to cool.

7.  Open the lid of the plate just enough to fit the loop in. Place the loop in the center of the second section and gently drag the loop one time into the third section. Lightly drag the tip of the loop from side to side in a back-and-forth motion to spread the inoculum to fill the third section. Replace the Petri Plate lid.

8.  Sterilize the loop in the Bacticinerator and allow it to cool.

9.  Incubate your Gram-Positive Stock Plate lid side down in a candle jar since some of the unknown Gram-Positive organisms are microaerophilic.

10.  Place the CNA plate lid side down in a candle jar or the class tray to be saved in the refrigerator in case your Gram-Positive stock culture does not grow.

MAKE THE GRAM-positive STOCK CULTURE Alternative streak plate

1. Label a TSA plate with your name, your unknown number, and the words “Gram Positive Stock”. Divide the plate into 3 sections by drawing a “T” on the plate. THIS WILL BE YOUR GRAM POSITIVE STOCK CULTURE. You will use this stock culture to inoculate all future biochemical tests performed on this unknown. TSA is a “non-inhibitory” media which allows the organism to produce enzymes and metabolize the ingredients in the media.

2.  Sterilize your inoculating loop in the Bacticinerator and allow it to cool thoroughly. Use the inoculating loop take ONE SINGLE ISOLATED colony from the CNA Agar.

3.  Using your non-dominant hand, open the lid of the plate just enough to fit the loop in.

4.  Inoculate the first section of the plate by touching the loop down on the surface of the agar and drawing a zig-zag streak to fill that first area of the plate. Replace the Petri plate lid.

5.  Sterilize your inoculating loop in the Bacticinerator and allow it to cool.

6.  Open the lid of the plate just enough to fit the loop in. Inoculate the second section of the plate by touching the loop down in the middle of the first section of the plate and draw a line toward the second section.  Continue to draw parallel lines to fill the second section. Replace the Petri plate lid.

7.  Sterilize your inoculating loop in the Bacticinerator and allow it to cool.

8.  Open the lid of the plate just enough to fit the loop in. Inoculate the third section of the plate by touching the loop down by touching down in the middle of the second section of the plate and draw a line toward the third section.  Continue to draw parallel lines to fill the third section. Replace the Petri plate lid.

9.  Sterilize your inoculating loop in the Bacticinerator and allow it to cool.

10.  Incubate your Gram-Positive Stock Plate lid side down in a candle jar since some of the unknown Gram-Positive organisms are microaerophilic.

11.  Place the CNA plate  lid side down in a candle jar or class tray to be saved in the refrigerator in case your Gram-Positive stock culture does not grow.

Day 3: Begin testing your unknown organisms

1.  As soon as you have your dichotomous keys graded and your organisms growing on stock cultures, you can begin to perform biochemical tests. YOU MUST PERFORM THE TESTS IN THE ORDER ON YOUR DICHOTOMOUS KEY.

2.  Use your stock culture to perform the first test listed on your dichotomous key. Label the test media with your name, test name, date, and whether the organism is your Gram- Negative or Gram-Positive unknown. Incubate the test until the next lab session.

* You can only inoculate ONE test/unknown/day unless you can determine the results of the biochemical test right away.

3.  Work on identifying both unknowns simultaneously. Organize your work so you do not mix up the tests. In your test tube rack, place the stock culture you are using in front of the biochemical test you are going to inoculate. Immediately label the test media with your name, date, media name and whether this is the Gram-Positive or Gram-Negative unknown.

4.  Once you have recorded the reactions and results, properly dispose of the test. Do not keep the biochemical tests after you have recorded the results.

5.  Once you have identified both unknowns, turn in your mixed unknown worksheet and dichotomous keys. After confirming your results with your instructor, dispose of all your test media, agar plates, and broths.

TESTS RESULTS OF GRAM-POSITIVE COCCI UNKNOWN

TEST RESULTS OF STAPHYLOCOCCUS AND MICROCOCCUS

TEST Staphylococcus aureus Staphylococcus epidermidis Micrococcus roseus Micrococcus luteus
Catalase Positive Positive Positive Positive
Glucose Fermentation Positive Positive Negative Negative
Coagulase Positive Negative Negative Negative
Salt Tolerance on Mannitol Salt Tolerance Positive Positive Negative Negative
Mannitol Fermentation on Mannitol salt Agar Positive Negative Negative Negative
Pigment Production (colony color) White White Rose-Coral Yellow
Nitrate Reduction (not done in this exercise) Positive Positive Positive Negative

 

TEST RESULTS OF ALPHA, BETA, AND GAMMA HEMOLYTIC STREPTOCOCCUS AND ENTEROCOCCUS 

TEST Streptococcus sanguinis Streptococcus pneumoniae Streptococcus pyogenes Group C
Streptococcus
Enterococcus
faecalis
Catalase Negative Negative Negative Negative Negative
Bile Esculin Negative Negative Negative Negative Positive
Hemolysis Alpha  Alpha Beta  Beta *Variable
Optochin sensitivity Resistant
(Negative)
Sensitive
(Positive)
Latex Agglutination Positive reaction with Group A antibodies Positive reaction with Group C antibodies

 

NOTE: “Variable” test result means the result is not consistent. Some strains of the species will exhibit gamma hemolysis whereas other strains will produce alpha or beta hemolysis.

TEST RESULTS OF GRAM-NEGATIVE Bacilli UNKNOWN

TESTS RESULTS OF GRAM-NEGATIVE UNKNOWN

TEST E.coli Klebsiella
aerogenes
Klebsiella
pneumoniae
Proteus mirabilis Salmonella Shigella Pseudomonas aeruginosa Alcaligenes faecalis
Oxidase Negative Negative Negative Negative Negative Negative Positive Positive
Glucose fermentation Positive Positive Positive Positive Positive Positive Negative Negative
Lactose fermentation Positive Positive Positive Negative Negative Negative Negative Negative
Sucrose fermentation Negative Positive Positive Negative Negative Negative Negative Negative
Nitrate reduction Positive Positive Positive Positive Positive Positive Positive Negative
Urease Negative Negative Positive Positive Negative Negative Not reliable Negative
Indole Positive Negative Negative Negative Negative Positive Not reliable Not reliable
MR Test Positive Negative Negative Positive Positive Positive Negative Negative
VP Test Negative Positive Positive Negative Negative Negative Negative Negative
Citrate Negative Positive Positive Positive Positive Negative Positive Not reliable
KIA H2S production Negative Negative Negative Positive Positive Negative Negative Negative
Motility Positive Positive Negative Positive Positive Negative Not reliable Negative

DISCOVERIES IN MICROBIOLOGY

Diagram of the hybridoma process for monoclonal antibody productionDR. CÉSAR MILSTEIN

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Laboratory Exercises in Microbiology Copyright © 2022 by Anne Mason M.S. and Jill Raymond Ph.D. is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License, except where otherwise noted.

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