STUDENT PROCEDURE WORKSHEET NO.

NAME: MARIA CHRISTMA B. UMBAÑA DATE: MAY 8, 2020

Differential Staining
Review Questions

1. What is the color of gram-positive organism?

The color of gram-positive organism is violet/purple.  This is due to retention of the

purple crystal violet stain in the thick peptidoglycan layer of the cell wall. 

2. What color does a gram-negative organism stain?

The color of the gram-negative organism is red/pin. This is because the structure of their
cell wall is unable to retain the crystal violet stain so are colored only by the safranin
counterstain. 

3. Gram stain result should include the gram reaction based on color, the organism’s cellular
shape, and any unique patterns ( for example, gram-positive cocci in clusters). What
differences can be noted between the two organisms (gram-positive and gram-negative)?

*Gram-positive bacteria have a thick peptidoglycan layer and no outer lipid membrane whilst
Gram-negative bacteria have a thin peptidoglycan layer and have an outer lipid membrane.
*Gram-positive bacteria have a distinctive purple appearance when observed under a light
microscope following Gram staining. Gram-negative bacteria appear a pale reddish color when
observed under a light microscope following Gram staining. 
*The thickness of the cell wall of gram-positive bacteria is 20 to 80 nanometres while the
thickness of the cell wall of gram-negative bacteria is 8 to 10 nanometres.
*In gram-positive bacteria, there is presence of teichoic acids while in gram-negative bacteria
there is absence of teichoic acids.
*In gram-positive bacteria, the outer membrane is absent while in gram-negative bacteria,the
outer membrane is present.
*The Porins of gram-positive bacteria is absent while in gram-negative bacteria it occurs in
outer membrane.
*The Mesosome in gram-positive bacteria is more prominent while in gram-negative bacteria, it
is less prominent.
*The morphology of gram-positive bacteria is cocci or spore-forming rods while in gram-
negative bacteria, it is non-spore forming rods.
*The flagella structure of gram-positive bacteria has 2 rings in basal body while in gram-
negative bacteria, it has 4 rings in basal body.
*The lipid content of gram-positive bacteria is very low while gram-negative bacteria has 20 to
30%.
*The Lipopolysaccharide of gram-positive bacteria is absent while in gram-negative bacteria,
there is present.
*The toxin produced by gram positive-bacteria is Exotoxins while gram-negative bacteria has
Endotoxins or Exotoxins.
*The resistance to antibiotic in gram-positive bacteria is more susceptible while gram-negative
bacteria is more resistant.

4. List the four reagents used in Gram stain and state the purpose of each reagent.

The first reagent used is the crystal violet where it is used as the primary stain/initial
stain. The next one is the gram’s Iodine where it is used as mordant. The third one is the 95%
alcohol or acetone alcohol that serves as the decolorizer and then the last one is the Safranin
where it is used as the counterstain/ secondary stain.

5. What would happen if the alcohol reagent was left on the Gram-stained slide too long?
Explain.

A decolorizer such as ethyl alcohol or acetone is added to the sample, which dehydrates

the peptidoglycan layer, shrinking and tightening it.  In the case of Gram-negative bacteria,
the alcohol/acetone mixture, being a lipid solvent, dissolves the outer membrane of the Gram-
negative cell wall. The decolorizer should stay on the slide for no more than 15 seconds. If the
decolorizer is left on too long, even gram positive cells will lose the crystal violet and
will stain red. It would create wrong results if this will be neglected. The purpose of this will not
properly because too much time being left on the slide will change the right structure of
organism.
 STUDENT PROCEDURE WORKSHEET NO.2

NAME: MARIA CHRISTMA B. UMBAÑA DATE: MAY 8, 2020

Preliminary Differentiation of Staphylococcus spp. from Streptococcus spp.

Review Questions

1. What is the principle behind the Catalase test?

The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and

water. The presence of the enzyme in a bacterial isolate is evident when a small inoculum is
introduced into hydrogen peroxide, and the rapid elaboration of oxygen bubbles occurs. The
lack of catalase is evident by a lack of or weak bubble production. The culture should not be
more than 24 hours old. Bacteria thereby protect themselves from the lethal effect of Hydrogen
peroxide which is accumulated as an end product of aerobic carbohydrate metabolism.

2. Staphylococci and Streptococci are differentiated by the catalase test. What constitute a
positive and a negative test?

The catalase test is used to differentiate staphylococci (catalase-positive) from

streptococci (catalase-negative). The enzyme, catalase, is produced by bacteria that respire
using oxygen, and protects them from the toxic by-products of oxygen metabolism. Catalase-
positive bacteria include strict aerobes as well as facultative anaerobes, although they all have
the ability to respire using oxygen as a terminal electron acceptor. Catalase-negative bacteria
may be anaerobes, or they may be facultative anaerobes that only ferment and do not respire
using oxygen as a terminal electron acceptor (ie. Streptococci).

3. What is the mode of action of catalase test?

The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and

water. The presence of the enzyme in a bacterial isolate is evident when a small inoculum is
introduced into hydrogen peroxide, and the rapid elaboration of oxygen bubbles occurs.
The catalase test tests for the presence of catalase, an enzyme that breaks down the harmful
substance hydrogen peroxide into water and oxygen. Bubbles are a positive result for the
presence of catalase. If no bubbles form, it is a negative result; this suggests that the
organism does not produce catalase.
NAME: MARIA CHRISTMA B. UMBAÑA DATE: MAY 8, 2020

CASE STUDY 1

A mother bring her 10-year- old daughter to the clinic; the child is complaining of a sore throat
and has a low grade fever (99.60F). the mother states that the child has had a runny nose and a
cough for the last few days. On examination, the physician notes that a child’s pharynx appears
red and that her tonsils are slightly swollen, no exudate is noted. Blood drawn for a complete
blood count (CBC), and a rapid strep test is sent to the microbiology laboratory. Laboratory data
follow:

Hemoglobin : Normal
White Blood count : Slightly elevated
Rapid Strep Test : Negative
Confirmation Culture : Negative for Group A ß-hemolytic streptococci

Multiple Choice Questions:

1. What is likely diagnosis for this patient’s condition?

A. Viral infection, the cause of the most cases of pharyngitis
B. Streptococcal pharyngitis, to be treated with antibiotics for 10 days
C. Acid fast organisms that would not be detected
D. No infection

2. What would be the follow -up testing for this patient?

A. No follow-up microbiology tests are needed
B. Have her return in 24 hours and repeat the CBC
C. Repeat the throat culture
D. Treat with antibiotics and then repeat the Rapid strep test

Critical Thinking Questions

1. What are the clinically relevant signs and symptoms in the case history?
2. What are the clinically relevant laboratory findings to support this diagnosis?
NAME _________________________________________ DATE _____________________

CASE STUDY 2

An 8-year-old man presents in the Emergency Department (ED) complaining of right sided chest
pain when he breathes and a productive cough. A sputum sample collected from the patient
revealed rust- colored sputum. He also states that his symptoms began abruptly with chills the
day before this visit to the ED, he had previously been healthy. Examination by the physician
identifies coarse breathing sounds in the right anterior chest. A chest radiograph shows a right-
upper-lobe infiltrate. The patient currently has a fever of 102 0F. Blood is drawn fa CBC. The
sputum sample is Gram-stained and cultured. Laboratory data follow:

Complete Blood Count

Hemoglobin : 14.5 g/dl (normal)
White Blood count : Elevated
Differential : 90% neutrophils (normal range 25% - 60%)

Sputum
Gram-stain : Gram-positive lancet shaped diplococci (cocci in pairs)
Culture Report : Streptococcus pneumoniae

The diagnosis is pneumonia caused by S. pneumoniae

Multiple Questions

1. The observation on which cells on the Gram-stained smear will assure the laboratory that
sputum specimen has been collected and tested?
A. Greater than 10 squamous epithelial cells per low-power field (10x)
B. Fewer than 10 squamous epithelial cells per low-power field (10x)
C. Greater than 10 columnar epithelial cells per low-power field (10x)
D. Greater than 10 gram-positive diplococci per low-power field (10x)

2. What characteristics of this patient would coincide with pneumonia caused by this organism?
A. the patient is otherwise healthy
B. the patient is most likely a smoker
C. the patient is an older adult
D. the patient is likely not providing all the details

Critical Thinking Questions

1. What are the clinically relevant signs and symptoms in the case history?
2. What are the clinically relevant laboratory findings to support this diagnosis?
NAME _________________________________________ DATE _____________________

CASE STUDY 3

A 20-year-old woman presents to her family practice physician for routine pelvic examination.
It has been several years since her last visit to this clinic, when she was diagnosed and treated
for a nongonococcal sexually transmitted disease (STD). She is sexually active with her fiancé
and has have sexual encounters with others in the past. She is currently considering the
potential for becoming pregnant. There are no apparent physical abnormalities, but the
physician decides to culture the cervical discharge for gonorrhea and to perform a DNA test.
Genprobe) for chlamydia and gonorrhea. A serum sample is also collected for HIV testing. All
laboratory tests are normal, with the exception for the test chlamydia, which is positive.
NOTE: Symptoms for Chlamydial infections include dysuria, and vaginal/urethral
discharge, symptoms similar to those of gonorrhea. Many infected patients exhibit no
symptoms of a chlamydial infection.

Multiple Choice Questions

1. Which of the following is the test of choice for the laboratory diagnosis of chlamydial
infection?
A. Nucleic acid amplification
B. Immunofluorescent antibody test
C. Enzyme Immunoassay (EIA)
D. Culture
2. If the patient is experiencing no symptoms, what would be the concern for the physician to
order the laboratory tests in the case?
A. To prevent unwanted transmission of organism to the fiancé
B. The organism present could lead to future infertility
C. The patient could develop more serious infection prior to symptoms
D. All the above statements are valid concerns in this case.

Critical Thinking
1. What are some of the significant clinical findings that would lead to the diagnosis of an STD?
2. What are some of the significant laboratory findings that would lead to the diagnosis of an
STD?
NAME _________________________________________ DATE _____________________

CASE STUDY 4

An 18 year-old college student complains of fever, chills, headache and vomiting. She presents
to the college health service ED, where she is examined. She appears lethargic, and her
temperature is 1020F. Blood is drawn for a CBC and culture, urine is collected for analysis, and a
serum chemistry profile is ordered. A lumbar puncture is performed, a cloudy cerebrospinal
fluid (CSF) is collected. Laboratory data follow:

Complete Blood Count

WBC count : 20.2 x 109 (normal range 5-10 x 109)
Differential : Marked neutrophilia with shift to immature forms (shift to
the left)

CSF Result
WBC count : 120 cells/mL with 95% neutrophils (reference value: 0-5
Lymphocytes
Glucose : 25 mg/dL (decreased compared with blood glucose value)
Protein : 150mg/dL (increased)

Other data
Gram stain : many neutrophils, gram-negative diplococci in pairs
Urinalysis : Increased protein, few RBC’s, few granular casts
Serum chemistries : within reference values
NOTE: Haemophilus influnzae (gram-positive coccobacillus) type B was the most common
cause of meningitisin children 1-6 years of age before the current vaccine became available.
Streptococcus pneumoniae (gram-positive diplococci) is a causative agent of meningitis in
adults. Neisseria meningitidis (gram-negative diplococci) is most frequently identified as the
causative organism for meningococcal infections in adults.

Multiple Choice Questions

1. From the patient’s history and laboratory results, all of the following findings in the blood
and CSF
substantiate a bacterial rather than a viral meningeal infection except which one?
A. Decreased CSF glucose
B. Increased WBC’s in CSF, with neutrophils predominating
C. Gram stain showing gram negative diplococci
D. Increased protein, few RBC’s, few granular casts in urine
2. What laboratory media would be required to isolate a pure culture of the suspected
infectious agent?
A. Sheep blood agar, McConkey agar, Eosin methylene blue
 B. Sheep blood agar, Thayer Martin agar, McConkey agar
C. Sheep blood agar, MaConkey agar, Chocolate agar
D. Sheep blood agar, SXT agar, Chocolate agar

Critical Thinking
1. What characteristics of the patient presented in the case history would provide clues as to
the potential
infectious agent?
2. Explain why the physician ordered the chemistry panel in conjunction with the other tests.