BIOD171 Final Lab Notebook
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Santa Fe College *
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171
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Biology
Date
Dec 6, 2023
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3
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Lab 9 Notebook
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Title: KD09 Unknown Pathogen Analysis
Objective:
To identify an unknown pathogen(s) based on the given assays developed
during this course. The goal is to correctly identify the 2 pathogens.
Procedure:
·
Test 1: Gram staining – gram positive (purple); gram negative (pink)
·
Test 2: MacConkey plate – only gram negative grows; grows lactose
fermenters
·
Test 3: Blood Agar Plate (BAP) – non-selective differential media contains
RBCs, important nutrient source, tryptic soy agar yeast + RBCs
·
Test 4: EMB plate – (grows for gram negative, dark red color, lactose
fermentation) selective and differential media; colonies turn green, partial lactose
fermentation; colonies turn pink, no color change in the bacteria, doesn’t produce
any enzymes that can break down lactose
·
Test 5: Catalase test (negative control had no bubbles) – determines the
presence or absence of catalase (an enzyme used to breakdown hydrogen
peroxide)
·
Test 6: Oxidase Test – (if oxidase negative it will be clear; if positive it will
turn purple) determine the presence or absence of cytochrome c activity in
bacteria
·
Test 7: Coagulase Test – (if it forms clumps it is positive; if it stays liquid
then negative) determines the presence of absence of coagulase which are
enzymes that play a role in the formation of blood clots
Notes:
Two samples from the hospital was brought to your lab for diagnosis. After culturing the
samples on LB agar plates another co-worker began the analysis. However, due to an
emergency your co-worker has left the remaining work to you. Before leaving your
co-worker informs you the 2 liquid cultures were accidently
combined
before the Gram
strain was started. However, the 2 cultures remain separated on LB plates for future
tests.
Results:
·
Test 1:
Gram staining
o
The cultures were mixed and viewed together. There is gram positive
bacteria (purple in color/round – coccus clusters) and gram negative
bacteria (pink in color/ bacillus rod-shaped)
·
Test 2:
MacConkey plate
– only gram negative grows; grows lactose
fermenters
o
Culture A: no growth
o
Culture B: did grow; the plate is mostly red with light clearing around the
streaking
·
Test 3:
Blood Agar Plate
– non-selective differential media
o
Culture A: growth; clearing around the streak – beta hemolysis
o
Culture B: growth; no clearing bacteria, white in color – gamma
hemolysis
·
Test 4:
EMB plate
(grows for gram negative, dark red color, lactose
fermentation)
o
Culture A: no growth (not able to ferment lactose)
o
Culture B: culture has a green metallic sheen (able to ferment)
·
Test 5:
Catalase Test
– (negative control had no bubbles) determines the
presence or absence of catalase
o
Culture A: catalase positive (bubbles)
o
Culture B: catalase positive (bubbles)
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Related Questions
procedure/s in performing aseptic transfer of bacterial cultures in (include illustration)
(2) agar slant culture to agar slant
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BONUS (15 points)
The fallowing series of dilution was prepared from a specimen to determine the number of bacteria.
There were 62 colonies on the agar plate prepared by transferring 0.3 ml from the tube number 4.
Calculate the dilution factors for each tube.
What is the cell concentration in the original specimen?
Calculate the total number of cells in tube number 2.
étv
A) F12
F9
F10
F7
%
&
%3D
delete
5
{
T
Y
J
K
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Cause-and-Effect Analysis: Given the conditions below, describe the effect on the staining procedure and explain briefly:
1. Excessive heat was applied during fixation
2. Low concentration of crystal violet used during gram staining
3. Excessive washing between steps
4. Insufficient decolorization
5. Excessive counterstaining
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Highlighted parts
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What are the concentrations of antibiotic applied in the test?
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First page is reference for number 4-7
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For laboratory exercise of Antibiotic susceptibility test in disk-diffusion method.
What does an ‘I’ result mean in disk diffusion susceptibility test?
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Topic: Preparation of Microbial Suspension
A Pure colony is needed for inoculation in sterile water, a small portion of culture media was included.
Effect:
Rationale:
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A 1.5-year-old child developed vomiting, diarrhea, and fever. Stool sample were inoculated into the Endo media. After 18 hours on the surface of the medium grew medium-sized, round, slightly convex red colonies with a metallic luster. The doctor suspected Escherichia coli.
1. Name the composition of the Endo agar media.
2. Describe the properties of bacterial colonies on the Endo media.
3. What purpose differential diagnostic Endo media used for?
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Sample Station 7
A. If a student performs a Gram stain on a Gram negative bacterium and
stops the stain technique after the iodine step and rinse, what color would
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microscope?
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Introduction
Discussion
References
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procedure/s in performing aseptic transfer of bacterial cultures in (include illustration)
(1) broth culture to broth
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Activity 2. Media Used in Isolating Coliforms
You are a group of microbiologists tasked to test the presence of coliforms in the newly built water station. You are
asked to isolate E. coli, Salmonella, and Shigella. Enumerate the media used for each step of isolating these
bacteria. Include pictures and references.
Bacteria
Enrichment
(Broth/Agar)
Presumptive test
(Broth/Agar)
Isolation Media
(Broth/Agar)
E. coli
Salmonella
Shigella
1. How can you confirm that E. coli, Salmonella, and Shigella are present in the newly built water
station? Explain.
2. What is the difference between nutrient broth and nutrient agar?
3. What is the importance of these steps (Enrichment, Isolation, and presumptive test) in isolating bacteria?
Conclusion about the process of isolating bacteria
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General Instructions:Choose 1 bacteria/diseases and fill out the details. An example of an answered template is also provided for your reference.
Causative Agent and Disease Profile for S. aureus
Template and Example
ITEM
MSM
PROFILE
MICROBIAL PROFILE
I
MICROORGANISM/CAUSATIVE AGENT
Staphylococcus aureus
A
GRAM REACTION
(+)
B
OXYGEN REQUIREMENT
Facultative Aerobes
C
SIZE
1.5 µm
D
SHAPE
Cocci in clusters
E
HABITAT
Normal flora of skin/anterior nares/pharynx
F
DISCOVERY
G
MICROSCOPIC IMAGE
II
DISEASE PROFILE
Scalded skin syndrome
A
DISEASE/S
Skin and Wound Infections
Scalded Skin Syndrome
Toxic Shock Syndrome
Food Poisoning Pneumonia
B
SYMPTOMS OF THE DISEASE
A high fever · Nausea and vomiting · A rash on your palms and soles that resembles a sunburn
C
INCUBATION PERIOD
2 and 4 hours (range 30 minutes to 8 hours)
D
MODE OF…
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Answer the following questions based on Kirby Bauer / MacFarland standards
1-0.5 McFarland standard solution is ideally used for inoculation of plated for Kirby Bauer test.
a)True
b)False
Principle of Kirby Bauer test is the diffusion of antibiotic agents in to Agar based media.
a) True
b) False
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Factors which can influence antibiotic susceptibility testing (size of zone inhibition) are_________________.
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The students of a Microbiology class were tasked to transfer or subculture a pure culture of Escherichia coli bacterium in five 7 mL nutrient broth and five petri dishes of nutrient agar with 20 mL capacity each. Based on the instruction bottles for nutrient broth and nutrient agar, preparation of the culture media is as follows.
Nutrient broth: 8 g/liter
Nutrient agar: 28 g/liter
Answer the following:
a. What is the weight in grams of nutrient broth?
b. What is the weight in grams of nutrient agar?
c. What is the distilled water in mL for nutrient broth?
d. What is the distilled water in mL for nutrient agar?
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Subculture and Colony Morphology Descriptions
Following identification of the Gram-negative isolate and Gram-positive isolate, you next subculture each onto fresh nutrient agar plates. Briefly describe the subculture process in three or so sentences and what this allowed you to achieve; follow this with colony morphology descriptions of each.
Description:
Isolate A – Describe:
Colony morphology:
Medium & incubation temperature:
Isolate B – Describe:
Colony morphology:
Medium & incubation temperature:
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Question:-
How to perform a bactericidal test bactericidal test of Dental Pulp Stem Cell (DPSC)?
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If both plates were accidently incubated at 37°C, how might interpretation of the results change? Explain.
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A class of 15 students (8 males and 7 females) will need to culture an unknown bacteria for a specific activity where 5 nutrient agar pates per female student and 3 agar slants per males student are needed to prepare.
agarplate: 25mL
Agar slant: 10mL
Broth tube: 8mL
Nutrient Agar:
Yeast extract: 2g/L
peptone: 5g/L
Sodium chloride: 5g/L
Agar powder: 15g/L
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Escherichia
coli
Mycobacterium
phlei
Bacillus
Micrococcus
subtilis
luteus
A
B
Figure 1: Agar Slant Cultures of Bacteria
(Gary E. Kaiser, Ph.D.- Professor of Microbiology)
. Observe and describe the colour of the slant cultures (A-D) in fig 1. (
. Define the following terms: pure culture, sterile medium, inoculum, aseptic
technique, and colony.
What is the name of the cultures that you used
. Where they gram negative or positive
Define the following terms: psychrophile, mesophile, thermophile, and
hyperthermophile.
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answer the following questions with book reference.
1. What is meant by a bacterial “colony” or colony-forming unit?
2. Why are colonies that develop on a heavily seeded plate smaller than those that appear on a sparely seeded plate?
3. What are some of the uses and advantages of the ff.:a. Agar plateb. Agar slantc. Broth culture
4. What are the advantages and limitations of studying bacteria by means of the Culture method?
5. Why is it desirable that most cultures be inspected after 15 to 18 hours of incubation?
6. Why should culture media after inoculation be incubated at an optimal temperature immediately?
7. Describe other types of aerobic jars and anaerobic methods.
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topic: BACTERIAL ENDOTOXINS TEST
Fill-in the possible effect of the procedure and give the rationale.
The sample solution prepared exceeded the MVD.
Effect:
Rationale:
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ISOLATE #1
Results for "Isolate #1"
***Please fill out the table****
1. Blood agar:
2. Nitrate Test:
Test
Results
Interpretation
Blood agar (type of hemolysis)
Nitrate test
3. Gelatinase Activity:
Gelatinase activity
Resistant or Susceptible?
Novobiocin Resistance
Zone Diameter:
4. Novobiocin Resistance:
5. Coagulase Test:
Coagulase (Sure-vue test)
1. Based on your results, what is the possible identification (Genus and species) of “Isolate #1"?
I 9 10 11 12 3 14
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- procedure/s in performing aseptic transfer of bacterial cultures in (include illustration) (2) agar slant culture to agar slantarrow_forwardBONUS (15 points) The fallowing series of dilution was prepared from a specimen to determine the number of bacteria. There were 62 colonies on the agar plate prepared by transferring 0.3 ml from the tube number 4. Calculate the dilution factors for each tube. What is the cell concentration in the original specimen? Calculate the total number of cells in tube number 2. étv A) F12 F9 F10 F7 % & %3D delete 5 { T Y J Karrow_forwardCause-and-Effect Analysis: Given the conditions below, describe the effect on the staining procedure and explain briefly: 1. Excessive heat was applied during fixation 2. Low concentration of crystal violet used during gram staining 3. Excessive washing between steps 4. Insufficient decolorization 5. Excessive counterstainingarrow_forward
- Hello, please read the attached Microbiology question and answer the question correctly. *If you correctly answer the question, I will provide a Thumbs Up to you. Thank you.arrow_forwardHighlighted partsarrow_forwardFor a laboratory exercise of antibiotic susceptibility test in a disk-diffusion method. What are the concentrations of antibiotic applied in the test?arrow_forward
- First page is reference for number 4-7arrow_forwardFor laboratory exercise of Antibiotic susceptibility test in disk-diffusion method. What does an ‘I’ result mean in disk diffusion susceptibility test?arrow_forwardTopic: Preparation of Microbial Suspension A Pure colony is needed for inoculation in sterile water, a small portion of culture media was included. Effect: Rationale:arrow_forward
- A 1.5-year-old child developed vomiting, diarrhea, and fever. Stool sample were inoculated into the Endo media. After 18 hours on the surface of the medium grew medium-sized, round, slightly convex red colonies with a metallic luster. The doctor suspected Escherichia coli. 1. Name the composition of the Endo agar media. 2. Describe the properties of bacterial colonies on the Endo media. 3. What purpose differential diagnostic Endo media used for?arrow_forwardSample Station 7 A. If a student performs a Gram stain on a Gram negative bacterium and stops the stain technique after the iodine step and rinse, what color would you predict the cells to be if you looked at it right now under the microscope?arrow_forwardQuestion:- Describe control strains used in the clinical microbiology laboratory and explain their maintenance in the laboratory. ( write BY WORD and all steps I need). Introduction Discussion Referencesarrow_forward
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Recommended textbooks for you
- Microbiology for Surgical Technologists (MindTap ...BiologyISBN:9781111306663Author:Margaret Rodriguez, Paul PricePublisher:Cengage Learning
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Biology
ISBN:9781111306663
Author:Margaret Rodriguez, Paul Price
Publisher:Cengage Learning