Transcript II_PROCARYOTIC CELL STRUCTURE

YOUR NAME: Dr. ?
YOUR eMAIL ADDRESS: ?
CASE: A Day at the Fair.
LEVEL 5
This PowerPoint tutorial explains is expected of your answers to
Level 5 of your case … It is too long to include in the “Example
Case” tutorial … Even at that, it is only an example and has been
pared down for this tutorial … The complete example is available
as the MSWord document
“06_EXAMPLE CASE STUDY_FAIRGROUND”
At the same time that you get the Level 4, you’ll also get the
“invitation” to teach a lab class to the 1st year med students …
… And you’ll be expected to completely discuss each type of
bacteriology growth media that you used 
YOUR NAME: Dr. ?
YOUR eMAIL ADDRESS: ?
CASE: A Day at the Fair.
LEVEL 5
Show-n-Tell Day in the Bact Lab!
For each specimen that was ordered (using the "Flamingham
General Hospital - Specimen Request Form" [Level 1 / Question
3]), which bacteriological growth media were actually inoculated?
The inoculation protocols, below, explain the media that are
always used; keep reading …
FLAMINGHAM GENERAL HOSPITAL
INOCULATION PROTOCOLS FOR THE ISOLATION OF
BACTERIAL PATHOGENS
This series of inoculation protocols was designed to ensure
that the typical bacterial pathogens, which might infect a particular
body site, are provided with the appropriate nutritional and
environmental conditions for growth. The Director of the
Microbiology Laboratory chose those tests and media because
they will allow the most efficient detection and/or isolation of the
typical bacterial pathogens that are usually associated with each
type of clinical specimen.
Observations of the pattern(s) of growth on all the media will allow
“Med Techs” [i.e. the skilled, although under-paid “Clinical
Laboratory Scientists”] and clinicians to make appropriate
decisions regarding the choice of any additional tests that will
ultimately lead to the correct identification of the pathogen.
Many bacteria require additional, special media and/or growth
conditions (special mixtures of atmosphere gases, temperature,
etc.) It is not economical to use these in a routine fashion.
A physician’s request (based on clinical history or other factors)
prompts for the special use of these.
Based upon his/her differential diagnosis, the practitioner may
order additional “differential” and “selective” media that are not
specified in these protocols
 Special media and additional tests can also be ordered
from the other clinical laboratories
(already done in Level 1/Questions 5 and 6, and there will be other
opportunities throughout the case.)
The routine inoculation protocols followed in FGH are below;
these protocols are a list of the media that according to your
hospital’s protocols MUST be used to isolate BACTERIAL
pathogens from EACH SPECIMEN that has been ordered.
EACH SPECIMEN MUST BE INOCULATED ONTO EACH OF
THE MEDIA AS “ASSIGNED” TO THAT TYPE OF SPECIMEN IN
THE TABLES, below.
Abbreviations of the Names of TESTS and GROWTH MEDIA
(Complete Descriptions of Each Medium Can Be Found in “Question 5C)
A Disk – Bacitracin-Impregnated Filter Paper Disk (To Identify Group A Strep)
- Blood Agar Incubated in an Anaerobic Chamber
BROTH – Brain Heart Infusion Broth
BLOOD – Agar Containing 5% (vol/vol) Sheep RBCs (Red Blood Cells)
CAMPY - Campylobacter Agar
CHOC - Chocolate Agar
EMB – Levine’s Eosine Methylene Blue Agar
HE - Hektoen Enteric Agar
- MacConkey Agar
/SORB – MacConkey Agar with Sorbitol
MTM - Modified Thayer Martin Agar
PEA - PhenylEthylene Alcohol Blood Agar
SELENITE - Selenite Broth
SMEAR – Smear of the Clinical Specimen on a Microscope Slide - for a Gram Stain
SS – Salmonella-Shigella Agar
THIO BROTH – Thioglycholate Broth - for Anaerobes
– Trypticase Soy Agar
V AGAR – Gardnerella vaginalis Agar – Agar Containing 5% (vol/vol) Horse RBCs
Note: Certain specimens [e.g. Blood, CSF, etc] should also be sent to the Chemistry and
Serology laboratories for additional, NECESSARY testing!
- Based on Level 1 / Question 3A in this example, the
ETIOLOGICAL DIAGNOSIS (“Specimen Request Form”), you
requested specimens of BLOOD and STOOL.
Specimen Request Form
X Blood
 CSF
 Catheter Tip
 Bone Marrow
 Dialysis Fluid
 Miscellaneous
Specimen
(e.g. Aspirate,
Abscess, Fluid,
Tissue, Wound,
Ulcer, etc.) *
* Specify source of
sample
EENT - Upper
Respiratory
 Eye
 Ear
 Nose,
Nasopharnyx
 Throat
Genital – Urinary
Tract
 Ulcer Swab
 Vaginal Swab
 Cervical Swab
 Midstream Urine
 Catheter Urine
 Urethral Swab
Lower Respiratory
Tract
 Sputum
 Alveolar/Bronchial
*
 Aspirate*
Gastro – Intestinal
Tract
X GI Stool
 Vomitis
INOCULATION PROTOCOLS FOR THE ISOLATION OF BACTERIAL PATHOGENS
SOURCE/TYPE OF SPECIMEN
MEDIUM and SPECIAL INSTRUCTIONS
NO SMEAR – This Specimen is NOT Routinely Gram Stained
BLOOD
BLOOD *
* An Automated (Anaerobic and Aerobic)
Blood Sample System, e.g. “BACTEC”
will be used in addition to these media
CHOC
MAC
ANA
Note: The Serology, Hematology, and Clinical Chemistry Laboratories, and perhaps
others should also analyze this specimen.
SMEAR - TO BE GRAM STAINED
CEREBRAL SPINAL FLUID
(CSF)*
* First CENTRIFUGE, Then Culture the
SEDIMENT
BLOOD
CHOC
MAC
THIO BROTH
Note: The Serology, Hematology, and Clinical Chemistry Laboratories, and perhaps
others should also analyze this specimen.
SMEAR - TO BE GRAM STAINED
CATHETER TIP
DIALYSIS FLUID
BONE MARROW
BLOOD
CHOC
THIO BROTH
SMEAR - TO BE GRAM STAINED
BLOOD
BLOOD - ANAEROBIC 37oC INCUBATOR
MISCELLANEOUS SPECIMEN *
* e.g. Wound, Abscess, Tissue Biopsy,
Synovial Fluid, Catheter Tip, Bone
Marrow, etc...
CHOC
PEA
PEA - ANAEROBIC 37oC INCUBATOR
MAC
ANA - If an Anaerobe Is Suspected
BHI - If Specimen Did Not Arrive in Transport Broth
SMEAR - TO BE GRAM STAINED
BLOOD
EYE
EAR
CHOC
MAC
MTM - EYE Culture only
NOSE
NASOPHARNYX
THROAT
Only upon physician's request: SMEAR - TO BE GRAM STAINED
BLOOD -CO2 INCUBATOR
CHOC - CO2 INCUBATOR
SMEAR - TO BE GRAM STAINED
SPUTUM
ALVEOLAR/BRONCHIAL *
LOWER RT ASPIRATE*
* First CENTRIFUGE, Then Culture the
SEDIMENT
* Request Special Medium and a second SMEAR if there is a suspicion of Tb
BLOOD
CHOC
MAC
If Midstream Urine - NO SMEAR – This Specimen is NOT Routinely Gram Stained
URINE - MIDSTREAM
URINE - CATHETER
If Catheter Urine - SMEAR - TO BE GRAM STAINED
BLOOD
MAC (or EMB)
SMEAR - TO BE GRAM STAINED
CERVICAL
VAGINAL
GENITAL ULCER
URETHRAL
BLOOD
CHOC
MTM
V AGAR
NO SMEAR – This Specimen is NOT Routinely Gram Stained
STOOL
VOMITIS
Upon Special Request - EXAMINATION FOR LYMPHOCYTES
BLOOD
MAC
HE
SS
CAMPY - Incubate at 42oC
SELENITE - Place Remaining Specimen on Swab in Selenite
Broth
BLOOD *
* An Automated
(Anaerobic and Aerobic)
Blood Sample System,
e.g. “BACTEC” will be
used in addition to these
media
STOOL
VOMITIS
NO SMEAR – This Specimen is NOT Routinely
Gram Stained
BLOOD
CHOC
MAC
ANA
Note: The Serology, Hematology, and Clinical
Chemistry Laboratories, and perhaps others should
also analyze this specimen.
NO SMEAR – This Specimen is NOT Routinely
Gram Stained
Upon Special Request - EXAMINATION FOR
LYMPHOCYTES
BLOOD
MAC
HE
SS
CAMPY - Incubate at 42oC
SELENITE - Place Remaining Specimen on Swab
in Selenite Broth
- So, for a BLOOD specimen, the lab must inoculate the patient's
sample on the following media: BLOOD, CHOC, MAC, ANA, and
no gram-stain will be performed (NO SMEAR)
- For the STOOL specimen, the lab must inoculate the patient's
sample on the following media: BLOOD, MAC, HE, SS, CAMPY,
SELENITE and no gram-stain will be performed (NO SMEAR)
- So overall, only the following media will be inoculated with the
patient's specimens: BLOOD, CHOC, MAC, ANA, HE, SS,
CAMPY, SELENITE and no gram-stain will be performed (NO
SMEAR)
So now answer the following question - For each specimen that was ordered
(using the "Flamingham General Hospital - Specimen Request Form" [Level
1/Question 3]), which bacteriological growth media were actually inoculated?
- Return to the series of "Bacteriology Inoculation Protocols" that are above and
Delete all of the table(s) regarding any specimen(s) that you did NOT request
using the "Flamingham General Hospital - Specimen Request Form"
- Then from the lists of tests/media associated with the specimens that were
Retained, highlight (or underline, whatever) the media in the list below that
were actually inoculated in the lab:
NO SMEAR
TSA
BLOOD
BLOOD - ANAEROBIC
37oC
CHOC
PEA
PEA - ANAEROBIC 37oC
MAC (or EMB)
ANA
BHI BROTH
THIO BROTH
MTM
V AGAR
HE
SS
SELENITE BROTH
CAMPY - 42oC
ENTEROCOCCUS
AGAR
Oh no!
You’re already so busy and don’t need another responsibility!
Dr. S. Kraut is ill. She is the president of the "Association of Fermenters for
the Next Millennia." A suspected food-borne illness associated with her "PickleOf-The-Month Club" will prevent her from her duties in the clinical labs. So
tomorrow, you have been assigned to fill in for her by guiding a group of 1st year
medical students through the clinical labs. You will have to lecture to your
students about the diagnostic process and will use your current patient as an
example.
During your introductory comments to the students, the lab techs will be
laying out all of the plates/broth tubes associated with your patient. You will
then discuss how each medium will hopefully provide valuable evidence
regarding the cause of your patient's illness. You had better be prepared!
So, based on your initial differential diagnosis, what do you expect to
observe on the plates? You must understand what will happen on every
medium for each of your "Most Likely Suspects." You do not wish to be
embarrassed in front of the students, and more importantly are concerned about
your patient. (You will actually get the most important of those results in either
level 2 to 4 of this case study.)
Oh no!
You’re already so busy and don’t need another responsibility!
In preparation for your prediction of which organisms will be on the
culture media, review what you have done and learned so far:
1) Initially evaluated the patient for the syndrome(s) that are associated with
his/her illness.
2) Considered all the "Most Likely Suspects" associated with the syndrome(s).
3) Ordered specimens that should shed light as to the cause of the illness.
4) Inoculated those specimens on the most appropriate media (using the
inoculation protocols, above) for bacterial pathogens most likely to be in the
patient's specimen(s).
FYI:
Types of Growth Media for the
Isolation and Identification of Bacteria from Specimens
GENERAL
PURPOSE MEDIA
Nutrient media (e.g. TSA) that support the growth of many microorganisms
ENRICHMENT
MEDIA
Supplemented nutrient media (e.g. TSA) to which necessary growth factors
(e.g. blood, serum, or extracts [e.g. yeast]) have been added to encourage
the growth of many fastidious bacteria
Uses: To isolate bacteria from CSF, pleural fluid, sputum, and wound
abscesses
SELECTIVE
MEDIA
DIFFERENTIAL
MEDIA
Culture media to which specific substances (e.g. dyes, chemicals, etc.)
have been added to favor the growth of one group of bacteria while
inhibiting the growth of other groups
Uses: To allow recovery of organisms in the presence of contaminating
microbiota
Culture media to which specific substances (e.g. dyes, specific sugars, etc.)
have been added to result in “diagnostically useful” growth or visible
changes in the media after incubation
Uses: To distinguish between different groups of bacteria based on their
biological characteristics - allows identification of organisms by specific
chemical reactions
GROWTH & APPEARANCE OF SUSPECTED PATHOGENS ON
ROUTINE BACTERIOLOGIC MEDIA
So now answer the following question - Demonstrate that the
specimens that were ordered and tests/media specified by the
“INOCULATION PROTOCOLS FOR THE ISOLATION OF
BACTERIAL PATHOGENS” are adequate, i.e. that those
tests/media are sufficient to allow the detection of all the usual
suspect bacteria that you listed in the DIFFERENTIAL
DIAGNOSIS from Level 1.
Based the answers below on –
your “enlightened” differential diagnosis
(comments/corrections on Level 1/Question 2 from the chief of infectious
diseases)
and the actual specimens that were ordered and were
worked-up in the bacteriology laboratory
(as listed at the beginning of level 2, perhaps not the specimens you had
originally considered in Level 1/Question 3).
For the following series of media table boxes, refer this info that
you prepared above,
NO SMEAR
ANA
HE
TSA
BHI BROTH
SS
BLOOD
THIO BROTH
SELENITE BROTH
BLOOD -
MTM
CAMPY - 42oC
ANAEROBIC 37oC
V AGAR
ENTEROCOCCUS
CHOC
PEA
PEA - ANAEROBIC
37oC
MAC (or EMB)
AGAR
Then:
- Delete each table box regarding any medium that is not required
by the FGH protocols
- Retain only the table box(es) regarding the media that must be
inoculated. Fill in those boxes as directed in the instructions.
Then for EACH Microbe and EACH Test/Medium - State your
Anticipated Observation and Explain why that observation might
occur 
Then for EACH Microbe and EACH Test/Medium - State your Anticipated
Observation and Explain why that observation might occur.
- This will require thought and effort; the info isn’t always easy to find. The
appropriate pages of the “Clinical Microbiology” chapter in an introductory
microbiology textbook (and/or other similar resources) should be consulted.
Search the internet using the genus and species names and/or use reference
texts available in many college libraries - e.g.:
KONEMANN’S Color Atlas and Textbook of Diagnostic Microbiology
or , RYAN, et al.’s SHERRIS- Medical Microbiology, An Introduction to
INFECTIOUS DISEASES
or MURRAY et al.’s The Manual of Clinical Microbiology
- But in some situations, you will not find the exact answers (unless
you extensively investigate several books and the internet).
You may have to try to “figure it out!”
So also read and consider the components and function of each
medium, and then base your answer on your knowledge of
culturing bacteria on artificial media; make an "educated guess!"
Cite the major references that you used to answer the questions
below.
? It’s your job to provide this info!
But here are some sites that others have found useful:
.
.
TEST
or
MEDIUM
BRIEF DESCRIPTION OF THE BACTERIOLOGICAL GROWTH
MEDIA
In addition to the specific ingredients discussed below, most media
contain salts [usually NaCl] and buffers to maintain the pH within the
range of neutrality [e.g. phosphate or “Tris”]
Genus species
of
”The Most Likely Causes
of Illness”
Please follow the correct
taxonomic style for writing
Genus species
Observation & Interpretation
Results of the Test or Growth Medium and
Interpretations of the Growth (Or Lack of Growth) on
Culture Media
TEST
or
MEDIUM
BRIEF DESCRIPTION OF THE BACTERIOLOGICAL GROWTH MEDIA
In addition to the specific ingredients discussed below, most media contain salts [usually NaCl] and buffers
to maintain the pH within the range of neutrality [e.g. phosphate or “Tris”]
Genus species
of
”The Most Likely
Causes of Illness”
Please follow the
correct taxonomic
style for writing Genus
species
SMEAR – Smear of the Clinical Specimen on a Microscope Slide - for a Gram Stain
GRAM-STAIN distinguishes between Gm- and Gm+ bacteria.
- Useful for identification of pathogens and determining effective antibiotic treatment
- Include the necessary information regarding a second type of SMEAR if there is a suspicion of Tb
SMEAR
”The Most Likely
Causes Of Illness”
(i.e. Bacteria being
“Considered” in the
Differential
Diagnosis)
Genus
species
TSA
(AGAR)
”The Most Likely
Causes Of Illness”
(i.e. Bacteria being
“Considered” in the
Differential
Diagnosis)
Genus
species
Observation & Interpretation
Results of the Test or Growth Medium and Interpretations of the Growth (Or Lack of Growth) on
Culture Media
Observation & Interpretation – What is the MICROSCOPIC appearance of the CELLS?
Gram+ or Gram- ... Cellular Arrangement and Morphology?
… Is it likely that the organism will be seen in a Gram-stained smear from a patient specimen?
Observation & Interpretation
TSA – Trypticase Soy Agar
ENRICHMENT MEDIUM - Trypticase Soy Agar – rich basal media containing on an acid hydrolysate of
CASEIN [a milk protein] and of SOYTONE [a soy bean protein extract]
Observation & Interpretation - What is the MACROSCOPIC appearance of the COLONIES?
… Does the microbe grow in this medium? … If yes, describe the Colony Size, Color, Texture, and any
other SELECTIVE or DIFFERENTIAL Characteristics that can be observed on the medium. … If no,
explain why it will not grow.
Observation & Interpretation
BLOOD
AGAR
BLOOD – TSA Agar Containing 5% (vol/vol) Sheep RBCs (Red Blood Cells)
ENRICHMENT MEDIUM - TSA (Trypticase Soy Agar) with the addition of 5%
[vol/vol] citrated sheep red blood cells (RBC) as an additional source of nutrition
DIFFERENTIAL MEDIUM showing HEMOLYSIS [lysis of the RBCs] -  [ALPHA] - partial hemolysis resulting in a greenish to brownish halo around a
colony
-  [BETA] - complete hemolysis resulting in a clearing effect around a colony
-  [GAMMA] - NO hemolysis – no change in the surrounding medium
If culture requires growth in an ANAEROBIC 37oC INCUBATOR, Explain Why.
”The Most Likely
Causes Of Illness”
(i.e. Bacteria being
“Considered” in the
Differential Diagnosis)
Observation & Interpretation - What is the MACROSCOPIC appearance
of the COLONIES?
… Does the microbe grow in this medium? … If yes, describe the Colony
Size, Color, Texture, and any other SELECTIVE or DIFFERENTIAL
Characteristics that can be observed on the medium. … If no, explain why it
will not grow.
Genus species
Observation & Interpretation
E. coli O157:H7 large, mucoid, gamma hemolysis
C. jejuni
?
CHOC - Chocolate Agar
ENRICHMENT MEDIUM - TSA (Trypticase Soy Agar) containing 5% [vol/vol] citrated sheep blood that
has been heated to 60oC to lyse the blood cells and release HEMIN and NAD [readily accessible sources
of additional sources of nutrition]
* Especially important for “FASTIDIOUS” microbes, i.e. those which are especially nutritionally
demanding]
If culture requires growth in a CO2 INCUBATOR, Explain Why.
CHOC
AGAR
”The Most Likely Causes Of
Illness”
(i.e. Bacteria being
“Considered” in the
Differential Diagnosis)
Observation & Interpretation - What is the MACROSCOPIC appearance of the COLONIES?
… Does the microbe grow in this medium? … If yes, describe the Colony Size, Color, Texture, and
any other SELECTIVE or DIFFERENTIAL Characteristics that can be observed on the medium. … If
no, explain why it will not grow.
Genus species
Observation & Interpretation
E. coli O157:H7
?
C. jejuni
?
PEA - PhenylEthylene Alcohol Blood Agar
ENRICHMENT MEDIUM - Blood Agar
SELECTIVE MEDIUM that contains 2.5% [v/v] PHENYLETHYLENE ALCOHOL which inhibits
growth of Gm- bacteria (permitting growth of Gm+)
DIFFERENTIAL MEDIUM that demonstrates HEMOLYSIS
If culture requires growth in an ANAEROBIC 37oC INCUBATOR, Explain Why.
”The Most Likely
Observation & Interpretation - What is the MACROSCOPIC appearance of the
Causes Of Illness”
COLONIES?
(i.e. Bacteria being
… Does the microbe grow in this medium? … If yes, describe the Colony Size, Color,
“Considered” in the
Texture, and any other SELECTIVE or DIFFERENTIAL Characteristics that can be
Differential Diagnosis)
observed on the medium. … If no, explain why it will not grow.
PEA
AGAR
Genus species
MAC
AGAR
Observation & Interpretation
MAC - MacConkey Agar
SELECTIVE MEDIUM - basal media containing BILE SALTS and CRYSTAL VIOLET to inhibit growth of Gm+
bacteria (permitting growth of enteric Gm-, the Enterobacteriaceae, and related Gm- bacilli)
DIFFERENTIAL MEDIUM that contains LACTOSE as the sole carbon source and neutral red dye to differentiate
LACTOSE FERMENTERS [various shades of red] from non-lactose-fermenting bacteria [colorless or transparent]
”The Most Likely Causes
Of Illness”
(i.e. Bacteria being
“Considered” in the
Differential Diagnosis)
Observation & Interpretation - What is the MACROSCOPIC appearance of the COLONIES?
… Does the microbe grow in this medium? … If yes, describe the Colony Size, Color, Texture, and any
other SELECTIVE or DIFFERENTIAL Characteristics that can be observed on the medium. … If no,
explain why it will not grow.
Genus species
Observation & Interpretation
E. coli O157:H7
?
C. jejuni
?
And so on for all the other media …
Thanx for not falling asleep!
 Note to the Doctor: There are certain human pathogens that will
continually reappear in your differential diagnoses.
The goal of this question is to make you understand how the
standard culture media function and how they facilitate the
diagnosis of bacterial infectious diseases, or aid in the diagnostic
process by "ruling-out" various bacteria as the cause of the
patient's illness.
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