5 - MICROBIOLOGY

Gram Stain

Differential stain to classify bacteria into two major groups: Gram positive and Gram negative, based on the color

Also on their morphology (cocci, bacilli or spirilli)

5-10

Crystal Violet

1

Gram Stain

1. Make sure the smear is properly air dried before doing the procedure

2. Turn on the heat plate and adjust the knob to increase the temperature. Turn off the heat planet when it reached its hottest temperature then place all the slides for heat fix for just _-_ seconds

3. Place the heat fixed slides in a cool surface and wait for it to cool down

4. Once cooled down, place all the slides into the staining rack

5. Flood the smear with _ _ (primary stain) and stand for _ minute

6. Gently wash the excess stain with tap water

Gram’s Iodine, 1

Acetone Alcohol, 5-10, 1-2

Safranin, 30

Gram Stain

1. Flood the smear with _ _ (mordant) for _ minute

2. Gently wash the excess reagent with tap water

3. Using _ _ as a decolorizer; flood the smear with acetone alcohol and allow it to stand for _-_ minutes (for thick smears) and _-_ minutes (for thin smears). Make sure when doing the decolorization process, check if there's still stain pouring out of the smear. If there is none anymore, wash the excess acetone alcohol with tap water

4. Flood the smear with _ (counterstain) and stand for _ seconds 

5. Gently wash the excess stain with tap water

6. Place the newly stained smears on the rack to air dry

Acid Fast Stain

A laboratory test that determines if a sample or specimen is infected with the bacteria that causes Tuberculosis and other acid-fast organisms

Carbol Fuchsin, 30

Acid Alcohol, 5-10, 1-2

Malachite Green, 1

Acid Fast Stain

Procedure

1. Same procedure in Gram stain from number 1 to 4

2. Flood the smear with _ _ (primary stain) and stand for _ minutes

3. Gently wash the excess stain with tap water

4. Using _ _ as decolorizer; flood the smear with acid alcohol and allow it to stand for _-_ minutes (for thick smears) and _-_ minutes (for thin smears). Make sure when doing the decolorization process, check if there’s still stain pouring out of the smear. If there is none anymore, gently wash the excess acid alcohol with tap water

5. Flood the smear with _ _ (counter stain) and stand for _ minute

6. Gently wash the excess stain with tap water

7. Place the newly stained smears on the rack to air dry

Group B Strep. (GBS) Screening Test

A prenatal test for ? is used to detect the presence of this bacteria in the vagina and/or rectum of pregnant women. ? can pass from pregnant woman to her fetus during labor. This is rare and happens to 1 to 2 babies out of 100 when the mother does not receive treatment with antibiotics during labor

3

3

2

3

3

15

Group B Strep. (GBS) Screening Test

1. Add _ drops of Extraction Buffer A

2. Add _ drops of Extraction Buffer B and mix the liquids thoroughly

3. Roll the swab against the side of the Extraction tube for _ minutes

4. Add _ drops of Extraction Buffer C

5. Twirl swab and mix well

6. Squeeze the swab totally and discard, cover the tip

7. Remove the test from the sealed pouch. Add _ drops of specimen to sample well

8. Wait for _ minutes

Influenza A & B Plus

• Rapid immunochromatographic test for the qualitative detection of Influenza A and B virus antigens from Nasopharyngeal swab, throat swab, and nasal aspirate samples

• An in vitro diagnostic test for the qualitative detection of influenza A and B nucleoprotein antigens in nasopharyngeal (NP) swab and nasal aspirate samples, using the rapid immunochromatographic method. The detection is based on the monoclonal antibodies specific for the nucleoprotein of either influenza virus A or B

• It is intended to aid in the rapid diagnosis of influenza A and B viral infection. Negative results should be confirmed by other methods, such as cell culture

3

15

Influenza A & B Plus

1. Specimen Preparation: Immediately insert the sampled swab into the extraction buffer tube. Rotate the swab at least ten times while pressing the tip against the bottom and sides of the tube to release the sample into the liquid. Let the swab soak for one minute.

2. Remove Swab: Squeeze the sides of the tube as you remove the swab to ensure as much liquid as possible is released from the swab tip. Discard the used swab into a biohazard bag.

3. Seal the Tube: Tightly screw the nozzle cap onto the extraction tube.

4. Apply to Test Cassette: Open the foil pouch and place the test cassette on a clean, flat surface. Unscrew the small white cap on the extraction tube that allows for dropwise dispensing. Hold the tube vertically and add _ drops of the liquid into the sample well (marked "S") on the test cassette.

5. Wait for Results: Set a timer for _ minutes. Do not read the result before 15 minutes or after 20 minutes, as this may lead to inaccurate results.

CDAT

A rapid, in-vitro diagnostic test that checks a stool sample for markers of Clostridium difficile (C. diff) infection

10

CDAT

Simultaneously detects the C. difficile glutamate dehydrogenase (GDH) antigen and its toxins (Toxin A and Toxin B) in a single test, providing a quick and user-friendly way to screen for infection, with results often available in about _ minutes

Glutamate Dehydrogenase (GDH)

CDAT

• _ _: An enzyme produced by all C. diff bacteria, both toxic and non-toxic

• _ _ and _ _: Toxins produced by certain strains of C. diff that cause severe diarrhea and colitis

3

chromatographic immunoassay

CDAT

• Procedure

  1. A stool sample is collected and mixed with a buffer solution

  2. The mixture is applied to the test strip (_ drops each well)

  3. The strip uses a _ _ to detect the GDH antigen and toxins

  4. Color lines appear on the test strip to indicate a positive or negative result for GDH, Toxin A, and/or Toxin B

BACT/Alert Virtuo for Blood Culture

Machines

For blood culture

Colorimetric

Machines

BACT/Alert Virtuo for Blood Culture

Principle

Anaerobic

Aerobic

10

1-3

Machines

BACT/Alert Virtuo for Blood Culture

• Sample:

  • Adult (_ – Orange & _ bottle – Green) – _ mL

  • Pedia (Peds plus – _-_ mL)

Gram stain & change in color

Machines

BACT/Alert Virtuo for Blood Culture

• Positive Result: (2) due to used up oxygen

Antibiotic Removing Device (ARD)

yellowish-brown

green

Machines

BACT/Alert Virtuo for Blood Culture

• Blood culture bottle containing a resin that removes antimicrobials (antibiotics) from the specimen

• Positive = _-_

• Negative = _

Biofire Filmarray (TORCH)

Machines

The system is designed to help physicians quickly identify a wide range of bacteria, viruses, and other pathogens for faster patient triage and treatment decisions

Nested Multiplex PCR

Machines

Biofire Filmarray (TORCH)

Principle

Respiratory Panel, 45

Pneumonia Plus Panel

GI Panel

Meningitis Encephalitis

Blood Culture / Identification Test

Machines

Biofire Filmarray (TORCH)

• 5 Panels:

  1. _ _

     • Viruses or bacteria

     • Spx: Nasopharyngeal swab

     • Running time: _ minutes

  2. _ _ _

     • Semi-quantitative

     • Antimicrobial resistance

     • Spx: Sputum or Bronchial Lavage

  3. _ _

     • Bacteria or parasite

     • Spx: Stool

  4. _ _

     • Bacteria, viruses, yeast

     • Spx: CSF

  5. _ _ / _ _

     • Gram positive, Gram negative, yeast

     • Spx: Positive blood culture bottle

Vitek MALDITOF-MS

Machines

The MS principle consists of ionizing chemical compounds to generate charged molecules and to measure their mass-to-charge ratio. Such molecular “signatures” can be used for rapid bacterial identification (ID) from isolated colonies

Parang binabasag yung organism (parang binabaril)

Mas mabilis = tataas na una

Bacteria, Yeast, Filamentous, Non-TB

Matrix Assisted Light Desorption Ionization – Time of Flight Mass Spectrometry

Machines

Vitek MALDITOF-MS

Principle

1-2

Machines

Vitek MALDITOF-MS

Running Time: _-_ hours

Vitek 2

Machines

• Automatic system for the identification and susceptibility testing of the most clinically important bacteria

• Gram Positive cocci only (18 hours incubation and dapat identified in gram stain)

• Can’t identify Haemophilus spp

Biochemical

MIC (specific) or Broth Microdilution Minimum Inhibitory Concentration

Machines

Vitek 2

• Principle:

• Identification – _

• Sensitivity – ?

GeneXpert

Machines

Automate and integrate sample purification, nucleic acid amplification, and detection of the target sequences in simple or complex samples using real-time PCR and RT-PCR always

Reverse Transcription Polymerase Chain Reaction (RTPCR)

Machines

GeneXpert

Principle

MTB-PCR, CT/NG (Chlamydia trachomatis, Neisseria gonorrhoea)

Rifampicin, MDR

Lowenstein-Jensen

Machines

GeneXpert

• Tests: (3)

• Can also test for the Susceptibility of _ (whether R or S) – screening for _

• Gold Standard for Detection of MTB: Culture (_-_ Agar)

BACTEC MGIT 960

Machines

Detects bacterial growth by measuring a change in fluorescence that indicates oxygen consumption. Initially, a fluorescent oxygen sensor in the liquid culture medium is quenched by dissolved oxygen, so there is little fluorescence. As bacteria grow and respire, they consume this oxygen and replace it with carbon dioxide, which lifts the quenching effect. This allows the sensor to fluoresce, with the intensity of fluorescence directly proportional to the amount of oxygen depleted, signaling bacterial growth.

Fluorescence

Machines

BACTEC MGIT 960

Principle

Salmonella and Shigella

Culture Media

Hektoen Agar

?

Corynebacterium diphtheriae

Culture Media

Tellurite Blood Agar

?

Legionella pneumophilia

Culture Media

Buffered Charcoal Yeast Agar

?

Vibrio cholerae

Culture Media

Thiosulfate Citrate Bile Salts Sucrose (TCBS)

?

Mycobacterium tuberculosis

Culture Media

Lowenstein-Jensen Agar

?

Corynebacterium spp.

Culture Media

Triple Sugar Iron (TSI)/Blood Agar Plate (BAP)

?

Haemophilus spp.

Culture Media

Chocolate Agar Plate (CAP)

?

Neisseria spp.

Culture Media

Modified Thayer Martin

?

E. coli (greenish metallic sheen)

Culture Media

Eosin Methylene Blue (EMB)

?

Enterobacteriaceae (Salmonella and Shigella)

Culture Media

Xylose Lysine Deoxycholate (XLD)

?

Bordetella

Culture Media

Bordet Gengou Agar

?

Clostridium difficile

Culture Media

Cycloserine Cefloxin Fructose Agar

?

Leptospira spp.

Culture Media

Fletcher Medium

?