BACTE

• SPECIMEN COLLECTION, MEDIA, AND METHODS

  Primary and differential media of choice for recovery of most fecal pathogens:

  1. Hektoen- salmonella and shigella
  2. MacConkey- lactose fermenters from non lactose
  3. CNA agar (colistin-nalidixic acid)- contains antibiotics that prohibit growth of gram-negative coliforms but not gram-positive cocci.
  4. Campy agar- contains the antibiotics cephalothin, trimethoprim, vancomycin, polymyxin B, and amphotericin B to prevent growth of Enterobacteriaceae, Pseudomonas spp., and fungi.
• TCBS Agar (Thiosulfate-citrate-bile-sucrose) agar

  • used to grow Vibrio cholerae, which appear as yellow colonies as a result of the use of both citrate and sucrose.

  APW (Alkaline peptone water)

  • used as an enrichment broth and should be subcultured to TCBS agar for further evaluation of Vibrio colonies.
• Colistin–nalidixic acid agar (CNA) is used primarily for the recovery of:

  • Staphylococcus aureus

  CNA agar inhibits the growth of gram-negative bacteria and is used to isolate gram-positive cocci from specimens. This medium is especially useful for stool and wound cultures because these may contain large numbers of gram-negative rods.
• These contains blood factors needed to support the growth of N. gonorrhoeae as well as antibiotics that prevent growth of normal genital flora:

  1. MTM (modified thayer-martin)
  2. New york city
  3. Martin-lewis agars

  Cultures must be incubated in 3%–7% CO2 at 35°C. Cultures should be held a minimum of 48 hours before being considered negative.
• Chocolate agar and modified Thayer–Martin agar are used for the recovery of:

  • Haemophilus spp. and N. gonorrhoeae, respectively
• CCFA (Cycloserine-cefoxitin-fructose agar) is used for the recovery of:

  • Clostridium difficile

  CCFA is used for recovery of C. difficile from stool cultures. Cycloserine and cefoxitin inhibit growth

  of gram-negative coliforms in the stool specimen.

  C. difficile ferments fructose, forming acid that, in the presence of neutral red, causes the colonies to become yellow
• Deoxycholate agar is useful for the isolation of:

  • Enterobacteriaceae

  DCA inhibits gram-positive organisms. N. gonorrhoeae and Neisseria meningitidis are too fastidious to grow on DCA. Citrate and deoxycholate salts inhibit growth of gram-positive bacteria. The media contain lactose and neutral red, allowing differentiation of lactose fermenters (pink colonies) from nonfermenters (colorless)
• Xylose lysine deoxycholate (XLD) agar is a highly selective medium used for the recovery of?

  • Enterobacteriaceae from gastrointestinal specimens
• XLD is differential for Shigella and Salmonella spp. The medium contains xylose, lactose, and sucrose, which are fermented by most normal intestinal coliforms producing yellow colonies. Shigella does not ferment the sugars and produces red colonies. Salmonella spp. ferment xylose; however, they also decarboxylate lysine in the medium, causing production of ammonia. Therefore, Salmonella first appear yellow but become red. Some Salmonella produce hydrogen sulfide from sodium thiosulfate and therefore appear as red colonies w/black centers.
• A sheep blood agar plate is used as a primary isolation medium when all of the following organisms are to be recovered from a wound specimen EXCEPT:

  1. β-Hemolytic streptococci and coagulase-positive staphylococci
  2. Haemophilus influenzae and Haemophilus parainfluenzae
  3. Proteus spp. and Escherichia coli
  4. Pseudomonas spp. and Acinetobacter spp.

  H. influenzae requires X and V factors, and H. parainfluenzae requires V factor; the primary isolation medium for Haemophilus is chocolate agar.
• Prereduced and vitamin K1-supplemented blood agar plates are recommended isolation media for:

  • Bacteroides, Peptostreptococcus, and Clostridium spp.
• Which procedure is appropriate for culture of genital specimens in order to recover Chlamydia spp.?

  • Inoculate cycloheximide-treated McCoy cells
• Specimens for virus culture should be transported in media containing:

  • Antibiotics and nutrient
• Media for transporting specimens for virus culture include Hanks balanced salt solution with bovine albumin, Stuart transport media, and Leibovitz–Emory media. Media used for transporting specimens for viral culture are similar to those for bacteria with the addition of a nutrient such as fetal calf serum or albumin and antibiotics. Specimens should be refrigerated after being placed in the transport

  media until the culture media can be inoculated.
• Cerebrospinal fluid (CSF) should be cultured immediately, but if delayed the specimen should be:

  • Incubated at 37°C and cultured as soon as possible
• Fastidious organisms such as Neisseria and Haemophilus frequently isolated from the CSF of patients with bacterial meningitis are preserved by placing the fluid in 3%–7% CO2 at 35°C–37°C (or at room temperature for no longer than 30 min), if the specimen cannot be cultured immediately
• The most sensitive method for the detection of β-lactamase in bacteria is by the use of:

  • Chromogenic cephalosporin
• The breakpoint of an antimicrobial drug refers to:

  • The level of drug that is achievable in serum

  The breakpoint refers to an antimicrobial concentration in the serum associated with optimal therapy using the customary dosing schedule. An organism is susceptible if the MIC is at or below the breakpoint.
• The following variables may change the results of an MIC:

  1. Inoculum size
  2. Incubation time
  3. Growth rate of the bacteria
• According to the Kirby–Bauer standard antimicrobial susceptibility testing method, what should be done when interpreting the zone size of a motile, swarming organism such as a Proteus species?

  • The swarming area should be ignored

  A thin film of growth appearing in the zone area of inhibition around the susceptibility disk should be ignored when swarming Proteus or other organisms are encountered. Discontinuous, poor growth or tiny colonies near the end of the zone should also be ignored
• Which class of antibiotics is used for the treatment of serious gram-negative infections as well as infections with Mycobacterium tuberculosis?

  • Aminoglycosides
• The aminoglycoside antibiotics are bactericidal agents that act by inhibiting protein synthesis. They show a low incidence of bacterial resistance but must be monitored carefully because at high doses they can cause ototoxicity and nephrotoxicity. The group includes amikacin, gentamicin, tobramycin, kanamycin, streptomycin, and spectinomycin. These drugs are usually administered intravenously or intramuscularly because they are poorly absorbed from the gastrointestinal tract.
• The medium best suited for the recovery of Yersinia enterocolitica from a patient with gastroenteritis:

  • Cefsulodin-Irgasan-Novobiocin (CIN) agar

  CIN agar inhibits the growth of many other organisms from the family Enterobacteriaceae. Yersinia spp. are also recovered from MacConkey and Salmonella-Shigella agars.
• A suspected case of plague requires the following procedures in order to confirm Yersinia pestis:

  1. Collection of multiple sets of blood cultures
  2. Incubation of blood cultures at both 28°C and 35°C
  3. Culture aspirates from bubos to MacConkey agar at room temperature
• SITUATION: Abdominal pain, fever, vomiting, and nausea prompted an elderly male to seek medical attention. A watery stool specimen producing no fecal leukocytes or erythrocytes was cultured and grew a predominance of gram-negative fermentative bacilli. The colonies were beta-hemolytic on blood agar and cream colored on MacConkey agar. The colonies were both oxidase and catalase positive. What is the most likely identification?

  • Aeromonas hydrophilia
• The oxidase positive test result rules out the members of the Enterobacteriaceae family. Colonies of Aeromonas hydrophilia and Plesiomonas spp. might be mistaken for Vibrio spp. since all three grow as clear colonies on MacConkey agar, are beta hemolytic on blood agar, and are oxidase positive.

  
• SITUATION: Several attendees of a medical conference in the Gulf coast area became ill after frequenting a seafood restaurant. A presumptive identification of Vibrio cholera was made after stool specimens from several subjects grew clear colonies on MacConkey agar and yellow colonies on TCBS agar. Which key tests would help eliminate Aeromonas and Plesiomonas spp.?

  • Mannitol fermentation, Na+ requirement
• All cocci are gram positive (+) except Neisseria, Branhamella, and Veillonella

  All bacilli are gram negative (-) except Mycobacteria, Corynebacteria, Clostridia, Bacillus, Erysipelothrix, Listeria, Rothia, Kurthia, and Lactobacillus
• 4 primary reagents used in gram staining:

  • crystal violet
  • gram's iodine (acts as a mordant)
  • decolorizer/acetone alcohol (95% ethanol)
  • safranin (Acts as the counter-stain)
• GRAM STAINING PROCEDURE

  1. Flood slide with crystal (or gentian) violet for 10 seconds, then wash with running tap water
  2. Subsequently flood with gram’s iodine for 10 seconds and wash with water
  3. Carefully decolorize it with 95% ethanol until thinnest parts of the smear are colorless and wash with water (this third step is the most critical and also the one most affected by technical variations in timing and reagents)
  4. Lastly, flood with safranin (pink color) for 10 seconds and wash with water afterwards. Afterwards, dry the slide via air drying or blotting using an absorbent paper
• Main principle behind gram staining:

  1. Gram-positive bacteria will retain the color of the primary dye (crystal violet) because of its very high link of peptidogylcan layer that absorbs the color of the crystal violet
  2. Gram-negative bacteria has only a thin layer of peptidoglycan which causes the crystal violet to be rinsed off upon the application of the decolorizing agent.
• Main principle of gram staining CONT

  1. Upon the application of the safranin (counter-stain), gram-negative bacteria absorbs the safranin while gram-positive bacteria remains purple due to the thick peptidoglycan layer’s absorbance towards the crystal violet iodine complex which is tough to remove during decolorization
  2. After gram staining: Gram-negative bacteria appears pink, while the gram-positive bacteria on the other hand, appears purple/violet in color.

  
• Results of gram staining

  • Gram-negative = PINK
  • Gram-positive = Dark purple / purple
• TWO KINDS OF ACID-FAST STAINING

  1. ZIEHL-NEELSEN STAINING

  ■ Employs heat-steaming to drive stain to the bacterial cell wall

  2. KINYOUN

  ■ Does not employ heat, instead it uses a detergent/tergitol to stain the bacterial cell wall
• STEPS IN ZIEHL-NEELSEN

  1. Application of the primary stain (carbolfuchsin) for 30 seconds
  2. Then heat-fix the cells by using a flame on the other side of the slide
  3. Decolorize using acid alcohol for 15-20 seconds

  • Note: If the bacteria has mycolic acid on its cell wall, it will retain the primary stain carbolfuchsin
  • If it has no mycolic acid, the carbolfuchsin will be rinsed off by the acid alcohol
• STEPS IN ZIEHL-NEELSEN

  4. Lastly, apply counterstain of methylene blue for 30 seconds then rinse the excess stain

  • Note: If it is an acid-fast organism, it will appear pink because it will retain carbolfuchsin due to the thick mycolic acid
  • Non acid-fast organisms will appear blue because it absorbed the counterstain methylene blue
• RESULTS OF ZIEHL-NEELSEN

  • Acid-fast organism = PINK
  • Non acid-fast = Blue
• SEROLOGICAL TESTS

  • Employed to know the bacterial infection
  • Principle: When bacteria infected a human, the human will respond by forming antibodies directed to these bacteria. If these antibodies are mixed with the same bacterium again, agglutination occurs
• SEROLOGICAL TEST

  • Detection of antibodies in patient’s serum can be done by mixing it with specific antigen that are commercially produced
  • Example:

  Preferred test for it correlates the condition of the patient.

  ○ Enzyme-linked immunosorbent assay (ELISA)

  ○ Antistreptolysin (ASO)

  ○ Western Blot
• General isolation media, also known as supportive media, support the growth of most nonfastidious bacteria. Examples include:

  • nutrient agar
  • trypticase soy agar
  • nutrient broth

  No growth advantage is given to any group of bacteria