1
Pathogens in Shellfish: selective agar use for presumptive identification
Leann Manley
Grades 7 – 12
Life science (Ecology, Biology, Microbiology)
Objectives
1) The students will be able to compare and contrast between the infectiousness (dose, symptoms and
victims) of Vibrio parahaemolyticus and Vibrio cholerae.
2) Students will be able to list 4 necessary environmental parameters for each of these two Vibrio
species.
3) The students will be able to describe in a short essay how these bacteria come to be in shellfish.
4) The students will be able to explain why the V.parahaemolyticus turns green on TCBS and the V.
cholerae turns yellow.
Background
Vibrio bacteria are naturally found in estuaries located in warmer climates. Especially in Florida,
V. parahaemolyticus is known for causing illnesses in those who enjoy raw oysters (among other
seafood). Especially immunocompromised individuals should heed the warnings posted in
oyster bars and not consume raw oysters (only post harvest treated or cooked oysters).
Vibrio cholerae
–
–
–
–
–
–
–
–
–
Gram negative
Creates an acid while fermenting sucrose which causes the colonies to turns yellow on
TCBS.
Other symptoms include rapid dehydration, rapid pulse, dry skin, tiredness, abdominal
cramps, nausea, and vomiting.
Optimal growth in a freshwater environment, but can survive in fresh, brackish or salt
water. Outbreaks usually follow plankton blooms.
Acquired through contaminated water or seafood
Ingested symptoms: explosive diarrhea. Suggested infective dose for V. cholera O1, 106
=1,000,000/g.
If given enough fluids it usually runs it course.
Vaccine, but it has a short-lived efficacy
2
Vibrio parahaemolyticus
–
–
–
–
–
–
–
–
–
–
Gram negative (less phospholipids in cell wall).
Halophile (in Florida estuaries) ~24 - 31 ppt.
Summer water temperatures 35 - 37ºC (95 – 98.6 ºF).
Symptoms: diarrhea > gastroenteritis > septicemia > death.
Ferments sucrose – turns green on TCBS
Acquired by ingesting seafood: shellfish, squid, mackerel, tuna, sardines, crab and shrimp
also through wounds.
Resolves in 3 days if not immunocompromised.
Following Hurricane Katrina, there were 22 Vibrio wound infections 3 of which were
caused by V. parahaemolyticus and 2 of these led to death.
Suggested infective dose – 106 = 1,000,000/g.
Vp adheres to plankton and then are ingested by shellfish as they filter feed.
Presumptive identification for these two Vibrio bacteria with TCBS agar.
TCBS
– Thiosulfate Citrate Bile salts Sucrose
– A selective agar for some Vibrio species
– Bile salts restrict a Gram + bacteria from growing (bile breaks down fats - phospholipids which are more readily available in Gram + bacteria)
– Sucrose fermented by parahaemolyticus = turns green on TCBS agar.
– Sucrose cannot be fermented by cholera = turns yellow on TCBS agar.
Additional Resources
Feel free to e mail me with ANY questions: [email protected]
I can point you in the right direction, and or directly answer your questions.
Fisher Scientific web site: http://www.fishersci.com/wps/portal/HOME?LBCID=14349421
Any microbiology textbook would serve as a great resource for streak plating, serial dilutions and
the methods. And Google is always there as well!
Other bacteria which grows on TCBS
V. alginolyticus yellow
V. cincinnatiensis yellow
V. damsela green
V. carchariae yellow/green
V. fluvialis yellow
V. furnissii yellow
V. metschnikovii yellow
V. vulnificus green
V. mimicus green
Aeromonas species yellow
Pseudomonas species blue/green*
Proteus species yellow/green*
Enterococcus species yellow
3
* The colonies are smaller than those produced by Vibrio species
Activity
**Ask if any students have shellfish allergies well in advance of this lab. Some shellfish allergies
are so severe the students should not be in the room during the lab.
**All students should wear eye protection, lab coats and gloves throughout this lab – even during
clean up! There should be no exceptions to any student for this request.
** No student at any time should put anything near their eyes or mouth during this lab.
Materials:
1)
2)
3)
4)
5)
6)
Blender
Incubator (optional)
Phosphate Buffer Solution (cheap to order from Fisher Scientific)
Oysters 3 - ?? as many as you want.
Shucking tool (a.k.a. shucker)
TCBS agar poured plates. (it is cheaper to buy the prepared powder, but you would need an
autoclave to sterilize it and most schools don’t have one). It’s up to you how many plates you want
each group to have. It could be as little as one or as many as three – there is a lot of room for
variation with this lab.
7) Q-tips (best to sterilize them in a UV goggles cabinet right before use). A fresh Q tip should be
used to streak each plate!
8) A guide sheet/instructions for students. This will depend on how you choose to set up the lab
based on your time and budget restraints.
Instructions:
Obtain ≥ 3 fresh oysters. The oysters must be fresh!! If they were frozen/treated in any way, at any
time, you will get no results!
Sample lab prep set up (upon purchasing the oysters fresh that day, within 1 hour do the
following):
1) Place 1 set (or 1 oyster) at room temp for 12 hrs. maximum (this variable should reveal
the most bacteria)
2) Place 1 set (or 1 oyster) in the refrigerator (till starting the lab) = the control.
3) Place 1 set (or 1 oyster) in the freezer (till starting the lab) (this variable should reveal
the least growth of bacteria).
Note: Very little homogenate is needed to streak a plate (a Q-tip swab). So, even one
homogenized oyster could service an entire class with several lab groups. There are many
ways to vary this lab to work in your classroom with your budget!
Warning: You should shuck the oysters, shucking is tough work and can be dangerous (use a
shucking tool and protective gloves!)
Instructions:
1) Shuck the oysters (1 – 3) from one variable, then place the meat in a blender with the
liquor included. Add an equal amount of Phosphate buffer (easy and cheap to order
through Fisher Scientific).
4
2) Homogenize for 60 seconds. Each variable should start with a clean blender! Repeat
these two steps for each variable)
At this point, if you want to do serial dilutions (which is kind of advanced for a regular high
school science class) to be able to count the number of bacteria I can give you instructions,
just e mail me at [email protected]
3) Streak the homogenate onto the TCBS plate with a Q-tip; dip the Q-tip into the
homogenate and zigzag it across the plate.(*Example below)
4) Incubate the plates (upside down) in an incubator for 24 hours, or leave them on the
counter for 48 hours.
5) Observe them and note what color the colonies are. Note also if nothing grew,
additionally note if there was more of one color than another. The students could
possibly count them, but it is not an accurate counting method for bacteria.
Note: This is a presumptive test – not confirmatory. The color change does not guarantee
that the colonies represent either of these bacteria, but the kids don’t know that! ☺
*Streaking plates: steps 1 – 4. A finished plate should look similar to #4. Use the same Q-tip throughout.
In order to make this lab inquiry based you could have them take swabs from areas around the school
(bathrooms, water fountains, cell phones, keyboards etc) and streak them onto TCBS and basic nutrient
agar and grow them up. Then have them observe the growth (or lack of) and write a conclusion on what
they found and why they think the results were as such.
Note: The TCBS should not have any growth as it contains salt.
5
Teacher’s Guide
V. parahaemolyticus on TCBS agar
V. cholera on TCBS agar
6
Assessments can vary from: 1) Have them write an essay conclusion of the results on a unit
exam. 2) A lab report 3) A prepared guided lab worksheet that they fill in as they work. 4)
Presenting their findings to the class (if the groups were split into different variables). 5) Have
them research another estuarine bacteria comparing it to V. cholerae or V. parahaemolyticus.
References
Information:
– Gooch, J., DePaola, A., Bowers, J., Marshall, D. 2002. Growth and survival of Vibrio
parahaemolyticus in postharvest American Oysters. J. Food Prot. 65(6):970-974.
– Oliver, J.D., Kaper, J.B. (1997) Vibrios. In Doyle, M.P., Beuchat, L.R., Montville, T.J.
Food Microbiology: Fundamentals and Frontiers. ASM Press. Washington D.C. pp. 228264
Pictures:
TCBS
www.bdj.co.jp/.../products/1f3pro00000rxoig.html
www.bio.dtu.dk/.../ugens_blog/weblog_trmu.aspx
Streak Plate
www.marine.csiro.au
Pathogens in Shellfish: selective
agar use for presumptive
identification
Objectives
• 1) The students will be able to compare and contrast
between the infectiousness (dose, symptoms and
victims) of Vibrio parahaemolyticus and Vibrio cholerae.
• 2) The students will be able to list 4 necessary
environmental parameters for each of these two Vibrio
species.
• 3) The students will be able to describe in a short essay
how these bacteria come to be in shellfish.
• 4) The students will be able to explain why the
parahaemolyticus turns green on TCBS and the V.
cholerae turns yellow.
Vibrio parahaemolyticus
–
–
–
–
–
Gram negative (less phospholipids in cell wall)
Halophile (estuaries/brackish)
Symptoms: diarrhea > gastroenteritis > septicemia
Ferments sucrose – turns green on TCBS
Seafood most often implicated includes shellfish, squid,
mackerel, tuna, sardines, crab and shrimp.
– Resolves in 3 days if not immunocompromised.
– Following Hurricane Katrina, there were 22 Vibrio wound
infections 3 of which were caused by V. parahaemolyticus and 2
of these led to death.
– Suggested infective dose – 106 = 1,000,000/g
Vibrio cholerae
– Gram negative
– Creates an acid while fermenting sucrose which causes the colonies to
turns yellow on TCBS.
– Other symptoms include rapid dehydration, rapid pulse, dry skin,
tiredness, abdominal cramps, nausea, and vomiting.
– Optimal growth in a freshwater environment, but can survive in fresh,
brackish or salt water. Outbreaks usually follow plankton blooms.
– Acquired through contaminated water or seafood
– Ingested symptoms: explosive diarrhea. Suggested infective dose for
V. cholera O1, 106 =1,000,000/g.
– If given enough fluids it usually runs it course.
– Vaccine, but it has a short-lived efficacy.
Cholera tent www.msf.org
V. cholera
jrubio.blogspot.com/
Presumptive Identification
TCBS
– Thiosulfate Citrate Bile salts Sucrose
– A selective agar for some Vibrio species
– Bile salts restrict a Gram + bacteria from growing
(bile breaks down fats which are more readily
available in Gram +)
– Sucrose which is fermented by V. parahaemolyticus
= turns green on TCBS agar.
– Sucrose fermentation in V. cholerae created and acid
= turns yellow on TCBS agar.
Vibrio parahaemolyticus on TCBS agar
Vibrio cholerae on TCBS agar
Activity
• Obtain ≥ 3 fresh oysters. The oysters must be fresh!! If
they were frozen/treated in any way, at any time, you will
get no results!
• Sample lab prep set up (upon purchasing the oysters
fresh that day, within 1 hour do the following):
– Place 1 set (or 1 oyster) at room temp for 12 hrs.
maximum (this variable should reveal the most
bacteria)
– Place 1 set (or 1 oyster) in the refrigerator (till starting
the lab) = the control).
– Place 1 set (or 1 oyster) in the freezer (till starting the
lab) (this variable should reveal the least growth of
bacteria).
• Note: Very little homogenate is needed to streak a
plate (a Q-tip swab). So, even one homogenized
oyster could service an entire class with several lab
groups. There are many ways to vary this lab to
work in your classroom with your budget!
• Warning: You should shuck the oysters, shucking is
tough work and can be dangerous (use a shucking
tool and protective gloves!)
Steps:
•
•
•
•
•
•
Shuck the oysters (1 – 3) from one variable, then place the meat
in a blender with the liquor included. Add an equal amount of
Phosphate buffer (easy and cheap to order through Fisher
Scientific).
Homogenize for 60 seconds. Each variable should start with a
clean blender! Repeat these two steps for each variable)
Streak the homogenate onto the TCBS plate with a Q-tip; dip the
Q-tip into the homogenate and zigzag it across the
plate.(*Example below)
Incubate the plates (upside down) in an incubator for 24 hours, or
leave them on the counter for 48 hours.
Observe them and note what color the colonies are. Note also if
nothing grew, additionally note if there was more of one color than
another. The students could possibly count them, but it is not an
accurate counting method for bacteria.
Note: This is a presumptive test – not confirmatory. The color
change does not guarantee that the colonies represent either of
these bacteria, but the kids don’t know that! ☺
Streak Plating
Other presumptive bacteria
•
•
•
•
•
•
•
•
•
•
•
•
•
Other bacteria which grows on TCBS
V. alginolyticus yellow
V. cincinnatiensis yellow
V. damsela green
V. carchariae yellow/green
V. fluvialis yellow
V. furnissii yellow
V. metschnikovii yellow
V. vulnificus green
V. mimicus green
Aeromonas species yellow
Pseudomonas species blue/green*
Proteus species yellow/green*
References
• Information
– Gooch, J., DePaola, A., Bowers, J., Marshall, D. 2002. Gowth and survival of
Vibrio parahaemolyticus in postharvest American Oysters. J. Food Prot.
65(6):970-974.
– Oliver, J.D., Kaper, J.B. (1997) Vibrios. In Doyle, M.P., Beuchat, L.R., Montville,
T.J. Food Microbiology: Fundamentals and Frontiers. ASM Press. Washington
D.C. pp. 228-264
– FDA – Online “The Bad Bug Book” Vibrio cholerae.
• Pictures
1) TCBS
www.bdj.co.jp/.../products/1f3pro00000rxoig.html
www.bio.dtu.dk/.../ugens_blog/weblog_trmu.aspx