9/11/21, 8:30 PM Printout: Fever and Abd Pain - Causes and Diagnosis

Fever and Abdominal Pain - Causes and Diagnosis

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                The patient presenting with fever and abdominal pain generates a broad differential diagnosis involving infections of the
gastrointestinal tract, solid organs of the abdominal cavity, gynecologic organs and referred pain from infections outside of the
abdominal cavity. The approach to diagnosis and antibiotic selection will follow that outlined in the chapter “Empiricism-Philosophy
and Practice” (Figure 1). The first step is to develop a problem list. From that problem list, a clinical differential diagnosis can be
generated. Based on the clinical diagnosis, a microbiologic differential diagnosis can then be created. The severity of illness, where
the infection was acquired (community or hospital based setting), underlying co-morbidities and drug allergies also need to be
accounted for. Finally, empiric antibiotic selection can occur only after careful consideration of the presumed microbiology, severity of
illness, and site of infection acquisition.
 
Figure 1: Approach to the Patient with Fever and Abdominal Pain

RUQ, right upper quadrant; RLQ, right lower quadrant; LLQ, Left lower quadrant; LUQ, left upper quadrant; AAS. Acute abdominal series; CT, computerized
tomography; PID, pelvic inflammatory disease
 
PROBLEM: Abdominal Pain
               The first division point is the general nature of the pain being either diffuse or localized. See Figures 1 and 2. Diffuse
abdominal pain raises the concern of a generalized peritonitis. Peritoneal irritation causes moderate to severe pain that is aggravated
by motion, forcing the patient to lay still. Bowel sounds are hypoactive or absent. There is voluntary and involuntary guarding and
rebound tenderness. Patients are often toxic appearing. Immune compromised patients, especially those on high dose
corticosteroids, warrant heightened vigilance since the anti-inflammatory effects of the corticosteroids may mask both fever and the
irritative signs of peritonitis.
               Pain localized to a specific area or quadrant of the abdomen may incriminate a specific organ infection involving the liver,
spleen, pancreas, kidney, bladder, colon, ovaries/salpinx or uterus (Table 2).
 
DIFFERENTIAL DIAGNOSIS
    Diffuse Abdominal Pain (Figures 1 and 2, Table 1)

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Diffuse abdominal pain may be due to bacterial infection of preexisting ascitic fluid, usually in the setting of hepatic
cirrhosis, referred to as primary or spontaneous bacterial peritonitis (SBP). Organ perforation with spillage of
gastrointestinal contents into the peritoneal cavity, referred to as secondary peritonitis, will also cause diffuse abdominal
pain though the onset is often more acute and the patient more toxic than in those with SBP. In the cirrhotic patient with
ascites presenting with diffuse abdominal pain, it is often challenging to differentiate primary from secondary peritonitis.
Acute abdominal series or computerized tomography (CT) may reveal free peritoneal air in the latter condition.
Secondary peritonitis is polymicrobic in nature while primary peritonitis is monomicrobic.
Table 1 outlines the clinical presentation, risk factors and diagnostic evaluation for diffuse abdominal pain (Problem # 1
from
Figure 1). Patients suspected of having SBP should undergo paracentesis. Fluid should be sent for cell count and
differential, protein content, lactate level, pH and Gram stain and culture. Some studies have suggested that the yield of
ascitic fluid culture may be increased by injecting the fluid into blood culture bottles for incubation. SBP is diagnosed by
the presence of bacteria on Gram stain or culture and/or a fluid white blood cell count > 250 cells/mm3. Supporting
evidence includes a protein > 3g/dl and a lactate > 25 mg/dl. Less commonly, diffuse abdominal pain may be referred into
the abdominal area from pneumonia, vertebral body infection or dermatomal Herpes zoster. Clostridium difficile
associated colitis should be considered in those who present with diarrhea, diffuse (or local) abdominal pain (often with
associated leukocytosis) and have recent exposure to antibiotics or have been recently hospitalized. Non-infectious
syndromes such as inflammatory bowel diseases, ischemic colitis, pancreatitis, polyarteritis nodosa (PAN), familial
Mediterranean fever (FMF), porphyria, sickle cell crisis and malignancies can present with fever and diffuse abdominal
pain.
Some infectious processes such as secondary peritonitis from gastrointestinal perforation are immediately life
threatening and require urgent diagnosis and surgical intervention. Others are more often subacute in nature (viral
hepatitis) and workup can proceed along at a less critical pace.
Table 1: Diagnosis of patients with diffuse abdominal pain
  Risk Factors Presentation Evaluation
Primary ·  Ascites due to cirrhosis, ·  Diffuse abdominal pain, ·  Blood cultures4
Peritonitis severe hypoalbuminemia fever, nausea, vomiting1 ·  AAS
from nephrotic syndrome,
CHF, malignancy ·  Leukocytosis3 ·  Abdominal CT   scan
·  CAPD catheter ·  Other signs and symptoms ·  Paracentesis2
·  VP shunts of hepatic failure5 ·  Liver enzymes7
·  Amylase, lipase
Secondary ·  Appendicitis ·  Acute onset of diffuse ·  AAS
Peritonitis ·  Cholecystitis abdominal pain, fever, · Abdominal CT scan
·  Diverticulitis nausea, vomiting · Liver enzymes7
·  Peptic ulcer disease ·  Abdomen rigid, hypoactive · Amylase, lipase
·  Abdominal injury or absent bowel sounds, · Exploratory laparotomy
·  Bowel obstruction guarding and rebound
·  Mesenteric ischemia tenderness  
·  Surgical anastomotic leak ·  Leukocytosis3
·  GU infections6
Lower ·
 Aspiration ·  Upper abdominal pain ·
 Blood cultures
Lobe ·
 Smoking ·  Cough, hypoxia may be ·  Chest x-ray
Pneumonia ·
 COPD present
·  Localized rales on chest
examination
CT, computerized tomography; CHF, congestive heart failure; COPD, chronic obstructive lung disease; GI, gastrointestinal; GU, genitourinary;
CAPD, continuous peritoneal dialysis catheter; VP, ventriculoperitoneal; AAS, acute abdominal series
1 Patients with cirrhosis and primary peritonitis may occasionally present without fever or abdominal pain. Consideration should be given to perform
paracentesis inpatients with ascites.
2 Ascitic fluid should be sent for white blood cell count and differential, protein, Gram stain and culture, lactate level and pH. Fluid may also be
inserted into a blood culture bottle for culture. The yield of ascitic fluid Gram stain and culture is poor. A negative test result does not exclude
spontaneous bacterial peritonitis (SBP). A fluid white blood cell count > 250 cells/mm3 is diagnostic of SBP.
3 Patients with overwhelming infections may have leukopenia and marked bandemia
4 Two sets should be obtained prior to the start of antibiotics. The yield of blood cultures in secondary peritonitis approaches 75%, while it is
substantially poorer in patients with SBP.
5 Encephalopathy, variceal bleeding
6 septic abortion, salpingitis, post partum endometritis
7 Liver enzymes; aspartate amino transferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, bilirubin

    Localized Abdominal Pain 

Localized abdominal pain may be due to many causes including 1) organ perforation with localized containment such as
diverticulitis, 2) infections of obstructed organs such as occurs in cholecystitis and appendicitis, 3) bacteremic spread
resulting in abscess formation in the kidney, liver or spleen and 4) spread of bacteria into otherwise sterile organs such
as the pancreas (infected pseudocyst or pancreatic abscess) or kidney (pyelonephritis). The differential diagnosis of

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localized abdominal pain can be approached by location of the pain: right upper quadrant (RUQ), left upper quadrant
(LUQ), right lower quadrant (RLQ) and left lower quadrant (LLQ). See Figure 2.
Table 2 outlines the clinical presentation, risk factors and diagnostic evaluation for localized abdominal pain (Problem # 2
from Figure 1). As with diffuse abdominal pain non-infectious illnesses such as those summarized above in addition to
acute alcoholic hepatitis, acute myocardial infarction, ectopic pregnancy and ruptured ovarian cyst may present with
localized abdominal symptoms that may mimic infection.  
Table 2: Diagnosis of Patients With Localized Abdominal Pain
  Risk Factors Presentation Evaluation
Hepatitis ·  Alcohol ingestion ·  RUQ pain ·  Liver enzymes7
·  IDU ·  Fever, nausea, vomiting ·  Serologic tests for viral
·  Ingestion of contaminated ·  Jaundice
food hepatitis3
·  Serologic tests for less common
causes as indicated4
Hepatic  ·
 Appendicitis, ·  RUQ and epigastric pain ·   Blood cultures
abscess Diverticulitis, ·  Fever, nausea, vomiting ·   Liver enzymes7
Cholecystitis ·  leukocytosis ·   RUQ ultrasound
· 
Bacteremia
·   CT scan
Cholecystitis1 ·  Gallstones · 
Postprandial RUQ and · 
Blood cultures
·  Trauma, burns epigastric pain · RUQ ultrasound5
· 
Fever, nausea, vomiting
·  Liver enzymes7
·  (+) Murphy’s sign2 ·  Amylase, lipase
·  Leukocytosis · 
HIDA scan
Cholangitis ·  Obstruction of the biliary ·  RUQ pain6 ·  Blood cultures
tree from gallstones, ·  Fever, nausea, vomiting ·  RUQ ultrasound
malignancy, surgery ·  Jaundice ·  Liver enzymes7
·  Leukocytosis ·  Amylase, lipase
Appendicitis ·       
Generally none ·     
 Periumbilical pain ·       
CT scan
·       
Foreign bodies migrating to RLQ
·       
Tumor ·  
 
Fever, nausea, vomiting
·       
Strictures ·       
Leukocytosis
·       
Parasitic infection8
Diverticulitis ·       
Diverticulosis ·       
LLQ pain9 ·       
Blood cultures
  ·       
Fever, nausea, vomiting ·       
CT scan
·       
Leukocytosis
Splenic ·       
Bacteremia ·       
LUQ pain referred to left ·       
CT scan
abscess shoulder
·       
Endocarditis ·       
CXR11
·       
Sickle cell disease ·       
Fever, nausea, vomiting
·       
IDU ·       
Leukocytosis
Colitis10 ·       
Contaminated food ·       
Diarrhea, ·       
Stool culture10
and water hematochezia ·       
Fecal leukocytes
·       
Antibiotics ·       
RLQ, LLQ pain ·       
Clostridium difficile toxin
·       
Fever assay
·       
Leukocytosis
Pelvic ·       
Young age and  sexual ·       
RLQ, LLQ pain ·       
Bimanual pelvic examination
Inflammatory
active12 ·       
Fever ·       
Pelvic ultrasound
Disease ·       
Leukocytosis ·       
CT scan
·       
New sexual partner
·       
Bacterial vaginosis ·       
Cervical culture for Neisseria
·       
IUD gonorrhea and Chlamydia
trachomatis
Endometritis ·       
Pregnancy13 ·       
Suprapubic pain ·       
Bimanual pelvic examination
·       
Fever
·       
Leukocytosis
Pancreatic ·       
Pancreatitis ·       
Periumbilical and back ·       
Blood cultures
abscess pain ·       
Liver enzymes7
·       
Fever ·       
Amylase, lipase
·       
Leukocytosis ·       
CT scan
Renal abscess ·       
Kidney stones ·       
Flank and back pain ·       
Blood cultures
·       
Ureteral obstruction ·       
Fever ·       
Urine culture
·       
DM ·       
Leukocytosis ·       
Renal ultrasound
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·       
Bacteremia ·       
CT scan
Pyelonephritis ·       
Kidney stones ·       
Flank and back pain ·       
Blood cultures
·       
Ureteral obstruction ·       
Fever, nausea, vomiting ·       
Urine culture
·       
DM ·       
Leukocytosis ·       
Renal ultrasound
IDU, injection drug use; RUQ, right upper quadrant; RLQ, right lower quadrant; LLQ, Left lower quadrant; LUQ, left upper quadrant; CT,
computerized tomography; DM, diabetes mellitus; CXR, chest x-ray; HIDA = hepatobiliary iminodiacetic acid, IUD, Intrauterine contraceptive
devices
1 95% due to gallstones. Acalculous cholecystitis can be seen after trauma, surgery, burns and in those with HIV infection, immune suppression and
DM
2 Murphy’s sign: inspiratory arrest during palpation of the RUQ. Named after John B. Murphy (1857- 1918), a Chicago, Illinois surgeon.
3 Hepatitis A virus IgG and IgM antibody, Hepatitis B surface antigen (HBsAg), Hepatitis B surface antibody (HBsAb), Hepatitis B core antibody
(HBcAb), Hepatitis C virus antibody.
4 IgM, IgG antibody for cytomegalovirus (CMV), monospot for Epstein Barr Virus (EBV) infection, antibody for human immunodeficiency virus
(HIV) infection
5 Thickened gallbladder wall, pericholecystic fluid, (+) sonographic Murphy’s Sign
6 The classic Charcot’s triad of RUQ pain, fever and jaundice is seen in less than 20% of patients.
7 Liver enzymes: aspartate amino transferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, bilirubin
8
Enterobius vermicularis, Ascaris lumbricoides, Strongyloides stercoralis
9 Pain may be RLQ or suprapubic depending on the position of the colon and location of the inflamed diverticula.
10 Including food borne bacterial colitis from Campylobacter, Salmonella, Shigella, Escherichia. Coli 015H7 and antibiotic related Clostridium
difficile.
11 May reveal left lower lobe atelectasis, effusion, elevated left hemidiaphragm
12  Pelvic inflammatory Diseases (PID) are often due to sexually transmitted pathogens such as Neisseria gonorrhoeae or Chlamydia trachomatis.
13 Seen more often with cesarian section, ruptured membranes for > 6 hours, multiple cervical examinations and chorioamnionitis.

Figure 2: Sequential Process for Thinking Through the Diagnosis, Microbiology and Therapy of a Patient with Fever and Abdominal Pain

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MICROBIOLOGY

                Decisions regarding antibiotic selection most often have to be made prior to the identification of the offending
pathogen(s). In many instances, microbial pathogens are never isolated or cultures are performed after antibiotics have
been initiated causing false negative results. In addition, an isolated pathogen may not fully represent the full
microbiology of an infection. For example, Escherichia coli
 may be the only isolate from a blood culture in a patient with
a diverticular abscess despite the polymicrobic nature of the infection. Therefore, the microbiology of an infection must be
based on the clinical diagnosis.
                Another important factor in thinking through the microbiology includes careful consideration of where the
patient acquired the infection. Infections acquired in the community are more often caused by less resistant aerobic gram
negative rods (GNR) such as E. coli, Klebsiella and Proteus spp.. Hospital acquired infections are more often caused by
more resistant GNR Enterobacter spp, Serratia spp, Morganella spp, Pseudomonas aeruginosa and vancomycin
resistant enterococci (VRE).
                Table 3 summarizes the microbiology of peritonitis causing diffuse abdominal pain. Primary bacterial peritonitis
is classically mono-bacterial. The bacteriology of secondary peritonitis depends on the site of the ruptured organ. Gastric
and upper small bowel flora often includes salivary organisms such as streptococci, lactobacillus and
candida spp.  The
distal jejunum, ileum and colon are polymicrobic including aerobic GNR, anaerobes such as bacteroides and clostridia
spp., and enterococci. . In the acutely ill patient with secondary peritonitis, it is often difficult to determine the origin of the
perforation prior to laparotomy. Therefore it is best to assume the worst microbial case scenario and assume the
perforation involves colonic bacteria.
                Table 4 summarizes the microbiology of localized intraabdominal infections. Those infections related to biliary
or gastrointestinal sources (diverticulitis, cholecystitis, appendicitis, pancreatic abscess) are polymicrobic. Pelvic
inflammatory disease (PID) is usually a community acquired sexually transmitted illness. Pyelonephritis and renal
abscesses are most often monobacterial. Abscesses in the liver can be polymicrobic (if originating from the gallbladder,
appendix or intestine) or monomicrobic (if due to bacteremic seeding) depending on the source, while splenic abscesses
are most often of bloodstream origin and monomicrobial.
 
Table 3: Microbiology of diffuse peritonitis
Clinical Diagnosis Community acquired Hospital acquired
Primary Peritonitis1 ·       
E. coli, Klebsiella pneumoniae, Proteus spp, ·       
Resistant  E. coli, Klebsiella spp, Proteus spp,
Enterobacter spp   Enterobacter spp    
OR OR
·       
S. pneumoniae        ·       
P. aeruginosa
OR
·       
Streptococci, enterococci
Secondary ·       
E. coli, Klebsiella spp, Proteus spp, ·       
Resistant  E. coli, Klebsiella spp, Proteus
Peritonitis2 Enterobacter spp    spp, Enterobacter spp, P. aeruginosa,
AND Serratia, Acinetobacter
·       
Enterococci                          AND
AND ·       
Enterococci including VRE    
·       
Anaerobes including Bacteroides, Clostridium, AND
Prevotella ·       
Anaerobes including Bacteroides,
Clostridium, Prevotella      
AND
·       
Candida spp.
1Primary peritonitis is most often monomicrobial. One third of patients have negative cultures  from paracentesis. Anaerobic bacteria are uncommon
and isolation should raise the concern of secondary peritonitis.
2 Secondary peritonitis is polymicrobic involving aerobic gram negative rods (GNR), enterococci and anaerobes.

 
Table 4: Microbiology of localized intra-abdominal infections
Clinical Diagnosis Community Acquired Hospital Acquired
Diverticulitis ·  Enteric GNR1  
  ·  Resistant  Enteric GNR2        
AND AND
·  Enterococci   
    ·  Enterococci including VRE  
  
AND  AND
·  Anaerobes3  ·  Anaerobes3
Appendicitis Same as diverticulitis Same as diverticulitis
Pancreatic abscess Same as diverticulitis Same as diverticulitis
Cholecystitis ·  Enteric GNR1 ·  Resistant  Enteric GNR2
Cholangitis
·  Anaerobes3,10  ·  Anaerobes3,10 
Hepatic abscess4 · 
Enteric GNR1    ·   Resistant  Enteric GNR2   
AND/OR AND/OR
·  Enterococci    
  ·   Enterococci including VRE   
AND/OR
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·  Anaerobes3    
  AND/OR
OR ·   Anaerobes3    
·  S. aureus, Streptococci, Candida, Yersinia5       OR
OR ·   S. aureus, Streptococci, Candida,
·  Entamoeba histolytica6
Splenic abscess7 ·  
S. aureus, Streptococci ·  MRSA, VRE
·   E. coli,
Klebsiella spp, Proteus spp, Enterobacter ·  Resistant  E. coli, Klebsiella spp, Proteus
spp spp, Enterobacter spp, P. aeruginosa,
Serratia, Acinetobacter
Colitis ·   
Campylobacter jejuni, Salmonella spp, Shigella ·   Clostridium difficile9
spp, E. coli  0157:H7, Vibrio parahaemolyticus,
Yersinia enterocolitica 8
·   Entamoeba histolytica6
·   Clostridium difficile9
Pelvic Inflammatory ·   Neisseria gonorrhoeae Not applicable
Disease ·   Chlamydia trachomatis
·   Enteric GNR1 and Anaerobes3
·   
Mycoplasma
Endometritis ·   Enteric GNR1 and Anaerobes3 ·   Resistant  Enteric GNR2 and Anaerobes3
·   Streptococcus agalactiae ·   Streptococcus agalactiae
·   Gardnerella vaginalis
Renal abscess ·  
E. coli, Proteus mirabilis, Klebsiella pneumoniae ·   Resistant  Enteric GNR2
·   S. aureus, streptococci ·   Enterococci including VRE
·   Candida spp
Pyelonephritis ·   E. coli, Proteus mirabilis, Klebsiella pneumoniae ·   Resistant  Enteric GNR2
·    Enterococci including VRE
·    Candida spp
Hepatitis ·  
Viral hepatitis A, B,C ·    CMV
GNR, Gram negative rod; CMV, cytomegalovirus; VRE, vancomycin resistant enterococci; MRSA, methicillin resistant Staphylococcus aureus
1
E. coli, Klebsiella spp, Proteus spp, Enterobacter spp
2 Resistant E. coli, Klebsiella spp, Proteus spp, Enterobacter spp, P. aeruginosa, Serratia, Acinetobacter
3
Bacteroides, Clostridium, Prevotella, anaerobic Streptococcus
4 Most often polymicrobic originating from infections of the hepatobiliary tree, appendicitis, diverticulitis.
5 May be monobacterial due to endocarditis or bacteremia.
6 Related to travel outside of the United States
7 Most often monobacterial related to bacteremia and endocarditis. 25% are polymicrobic.
8 Foodborne
9 Related to antibiotic usage.
10 Less often isolated except in patients with biliary-intestinal anastomosis.

DIAGNOSIS
               In the absence of physical findings of diffuse peritonitis, diagnostic imaging with either computed tomography (CT) or
ultrasound should be routinely performed in seriously ill patients with intra-abdominal infection. The urgency of investigation is
dictated by the degree of hemodynamic instability present. Most patients should be evaluated within hours of clinical diagnosis. This
initial imaging study has become central to therapeutic decision-making since interventional radiology has replaced operative
treatment for many localized processes, including diverticular abscesses. Double contrast CT is the single best modality for fully
evaluating the extent of disease in most situations. Ultrasound is also quite versatile and has the added advantage of being portable,
thus allowing certain procedures to be performed in the ICU. However, ultrasonography is limited by bowel gas, body habitus, and a
lower sensitivity for retroperitoneal processes or parenchymal infection. Usually the choice of modality is based on the experience
and preference of the interventional radiologist.

The Gram Stain

 
               The Gram stain is an empirical method of differentiating bacterial species into two large groups (Gram-positive
and Gram-negative) based on the chemical and physical properties of their cell walls. The method is named after its
inventor, the Danish scientist Hans Christian Joachim Gram (1853 – 1938), who formatted the technique in 1884.
               Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50-90% of cell wall), which
stain purple and Gram-negative bacteria have a thinner layer (10% of cell wall), which stain pink.
               Gram-negative bacteria also have an additional outer membrane which contains lipids, and is separated from the
cell wall by the periplasmic space. There are four basic steps of the Gram stain, which include applying a primary stain
crystal violet to a heat-fixed smear of a bacterial culture or specimen, followed by the addition of a mordant (Gram's
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iodine), rapid decolorization with alcohol or acetone and counterstaining with safranin. The Gram stain is the most
common staining procedure in clinical microbiology. This tool can aid the physician in determining if infection is present,
a possible etiology and a guide to choosing the appropriate antibiotic in just 15 minutes.
 
The Gram Positive Cell Wall
              The Gram positive cell wall The Gram positive cell wall is characterized by the presence of a very thick
peptidoglycan layer, which is responsible for the retention of the crystal violet dyes during the Gram staining procedure.
Imbedded in the Gram positive cell wall are polyalcohols called teichoic acids which are lipid-linked to form lipoteichoic
acids. Because lipoteichoic acids are covalently linked to lipids within the cytoplasmic membrane they are responsible for
linking the peptidoglycan to the cytoplasmic membrane. Teichoic acids give the Gram positive cell wall an overall
negative charge due to the presence of phosphodiester bonds between teichoic acids monomers.
The Gram Negative Cell Wall
              The Gram negative cell wall Unlike the Gram positive cell wall, the Gram negative cell wall contains a thin
peptidoglycan layer adjacent to the cytoplasmic membrane, which is responsible for the cell wall's inability to retain the
crystal violet stain upon decolorization with ethanol during Gram staining. In addition to the peptidoglycan layer, the
Gram negative cell wall also contains an additional outer membrane composed by phospholipids and lipopolysaccharides.

Gram Staining Tips

By Jack D. Rihs
1. Slide preparation
               Purulent material should be selected whenever possible.
               Apply the sample evenly and thinly to the slide. Smears that are too thick will be difficult to decolorize and
imposable to read.
               Do not cover the entire slide with the sample. This will make handling difficult and areas may be missed during
decolorization. An area the size of a nickel usually is adequate.
2. Slide fixation
               The material must be fixed to the slide to prevent it from washing off during staining. This can be done by
quickly passing the slide over a gentle flame (the slide should not become to hot to touch) or on a slide warmer.
Overheating may alter cell morphology or cause organisms to decolorize more quickly.
               An alternative and superior method of fixation is to flood the slide with methanol for 1 minute. Methanol fixation
prevents liquid specimens from washing off the slide better than heat fixing, preserves blood cell morphology and results
in a clearer background.
3. Staining
               Flood the entire slide when crystal violet, iodine and safranin are applied. This will ensure that all areas are
stained evenly.
4. Decolorization
               The critical step of the Gram staining procedure is the decolorization step. Hold the slide in a tilted downward
position and allow the decolorizer to flow over the smear. Be careful not to miss any portion of the smear. Usually a few
seconds will suffice.
               95% ethanol will decolorize slower than acetone/alcohol, than does acetone.
5. Reading the Gram stain
               Begin reviewing the slide using the 10x objective. This will allow you to focus quickly and to look for areas of
purulence. The 100x oil immersion lens is essential for viewing individual bacteria.
               When reviewing a Gram stain, look at many microscopic fields and in different areas of the slide.
               The presence of many squamous epithelial cells in the smear usually indicates a poorly collected sample that will
contain normal flora, whereas, the presence of polymorphonuclear leukocytes and no squamous epithelial cells indicates a
good sample and an inflammatory process.
               Some specimens on preparation will have areas on the slide that are thicker than others. This can result in areas
that are under decolorized, thus organisms that are truly Gram-negative will appear Gram-positive. Microscopic fields that
contain crystal violet precipitant, or PMNs, macrophages or epithelial cells that are staining purple are areas that are under
decolorized.
               Gram positive bacteria will usually stand out easily against the pinkish background. Gram negative organisms
because of their lower contrast can be missed, particularly in thicker smears. Haemophilus, Bacteroides and
Fusobacterium are Gram-negative bacteria
that are often overlooked due to their size, pleomorphic morphology or faintly
staining qualities.

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