In Vitro Phagocytosis: Principle, Materials and Procedure

Principle

A drop of whole blood is mixed with a drop of a bacterial culture and incubated at room temperature to demonstrate engulfment of bacteria by leukocytes.

Materials

• Test tubes, 12 75 mm 
• Broth culture of Staphylococcus epidermidis 
• Lancets for finger puncture 
• Heparinized microhematocrit tubes 
• Microscope slides 
• Wright stain

Procedure

1. Take two 12 75 mm test tubes and label one 0 minutes and the other 5 minutes. If there is enough blood available, a 10-minute tube can also be set up.
2. Do a finger puncture and fill a heparinized microhematocrit tube about three-quarters full of blood. Note that blood drawn in an EDTA tube within the last 10 minutes can also be used.
3. Using a black rubber bulb, expel one drop of blood into each labeled test tube.
4. Add one drop of Staphylococcus epidermidis culture to each tube, using a disposable Pasteur pipette. The culture should be no more than a 0.5 McFarland standard in concentration. Dilute in an additional broth tube or in sterile saline if necessary.
5. Shake the tubes to mix, and make a blood film of the 0 tube immediately. Let the other tubes incubate for 5 minutes and 10 minutes, respectively, at room temperature before making blood films of them. 

Method for Making Blood Smears

1. Obtain two clean glass slides, one of which will be used as a spreader slide.
2. With a Pasteur pipette, carefully place a small drop of blood at one end of a microscope slide.
3. Holding the spreader slide with the thumb and forefinger, place the spreader slide slightly in front of the drop of blood on the other slide, maintaining a 25-degree angle between the slides.
4. Move the spreader slide back toward the drop of blood. As soon as the slide comes in contact with the drop of blood, the blood will start to spread along the edge.
5. Keeping the spreader slide at a 25-degree angle, push it rapidly over the length of the slide. There should be a feathered edge on the end of the smear. 

Wright Stain

1. Allow the blood smear to air dry.
2. Stain according to typical laboratory protocol for staining blood smears.
3. Blot the back of the slides to remove excess stain and let them air dry.
4. Use immersion oil and look for engulfment.

Interpretation/Analysis

These blood smears will be more watery than usual due to the addition of the broth culture with bacteria; therefore, it may be difficult to get a blood smear with a feathered edge. The feathered edge is not essential, as long as the blood cells are spread out on the slide. There should be a noticeable difference between the 0- and the 5-minute slide. The 0-minute slide will probably not show much engulfment, but bacteria may be seen in contact with leukocytes. The 5-minute slide should show bacteria within the cell as small purple dots. Neutrophils will be the predominant phagocytic cells, but an occasional monocyte may be seen. If lymphocytes are the only white blood cell seen, the bacterial suspension was too heavy, and the phagocytic cells destroyed themselves in attempting to engulf the bacteria present.

Bacillus anthracis- Characteristics and Morphology

image courtesy of textbookofbacteriology.net

Bacillus species are large motile, facultative anaerobic, gram positive bacilli with a central spore. The spore is quite resistant to extreme conditions and can survive in nature for prolonged periods of time. 

B. anthracis is non-motile and in Gram stain is often seen in chains. The virulent forms of B. anthracis is more likely to be surrounded by a capsule. The organism can be cultured as large colonies on blood agar plates within 24 hours, often resembling a "Medusa head" (irregular appearance to the colony with swirling projections).

BACTEC MGIT 960 System- Principle, Manual and Brochure

The BACTEC MGIT 960 Mycobacterial Detection System is the world's first automated system for high-volume mycobacteria growth, detection and susceptibility testing from Becton Dickinson (BD). It is non-radiometric and uses BD BBL Mycobacteria Growth Indicator Tube (MGIT) media incubated at 37°C and patented sensors, making efficient use of advanced fluorometric technology which permits highly accurate detection of O2 consumption without sharps. Automated quality control is performed continuously to ensure precise and reliable operation. Results are provided as positive/negative and numerical Growth Units . The instrument scans the MGIT every 60 minutes to detect positive tubes as indicated by an increase fluorescence.

Mycobacterium tuberculosis DNA Fingerprinting from Epidemiologically Linked Case Pairs

image courtesy of blogs.scientificamerican.com

Authors: Bennett, Diane E.; Onorato, Ida M.; Ellis, Barbara A.; Crawford, Jack T.; Schable, Barbara; Byers, Robert; Kammerer, J. Steve; Braden, Christopher R.;

Publisher: CDC Open Access

Article source: Emerging Infectious Diseases. 8(11):1124-1129

Aspergillus Species Identification in Clinical Setting

Authors: S.A. Balajee, J. Houbraken, P.E. Verweij, S-B. Hong, T. Yaghuchi, J. Varga and R.A. Samson

Publisher: www.studiesinmycology.org

Article type: Journal Article

Abstract:

Multiple recent studies have demonstrated the limited utility of morphological methods used singly for species identification of clinically

BD Bactec FX- Fully Automated Blood Culture System Analyzer

image courtesy of www.medibio.tn

The BD BACTEC FX, latest among the bactec blood culture system, is a fully automated microbiology growth and detection system designed to detect microbial growth from blood specimens. It uses fluorescent technology with an exceptional performance, improving workflow efficiencies from specimen collection to actionable results. 

Advance Features:

Blood culture testing is one of the most important functions of the microbiology laboratory as clinicians rely on this information to aid in the diagnosis of bacteremia and fungemia. The BACTEC FX System builds on the proven history of previous  BACTEC instrumentation; it goes much further with exciting innovations such as: Vial-activated workflow, advanced ergonomics, remote alarms and blood culture

In Vitro Phagocytosis Test: Principle, Procedure and Interpretation

Principle:

A drop of whole blood is mixed with a drop of a bacterial culture and incubated at room temperature to demonstrate engulfment of bacteria by leukocytes.

Enterobacteriaceae and Non- Fermenters Study Guide- Microbiology

This study companion guide focuses mainly on enterobacteriaceae and some non- fermenters such as Pseudomonas aeruginosa and Burkholderia cepacia. 

Enterobacteriaceae is a large family of Gram-negative bacteria that includes, along with many harmless symbionts, many of the more familiar pathogens, such as Salmonella, Escherichia coli, Yersinia pestis, Klebsiella and Shigella. Other disease-causing bacteria in this family include Proteus, Enterobacter, Serratia, and Citrobacter. This family is the only representative in the order Enterobacteriales of the class Gammaproteobacteria in the phylum Proteobacteria.

IMViC Reaction: Knowing Your Coliforms

The IMViC tests are a group of different biochemical tests used in diagnostic clinical microbiology testing to identify an organism in the coliform (Enterobacteriaceae)  group. A coliform is a gram negative, aerobic or facultative anaerobic bacilli which produces gas from lactose within 48 hours of incubation. The presence of some coliforms indicate fecal contamination of the sample.

Except for the lowercase letter "i" is added for ease of pronunciation, each of the letters in "IMViC" stands for one of these biochemical tests.

• "I" is for Indole test;
• "M" is for Methyl red test;
• "V" is for Voges-Proskauer test, and
• "C" is for Citrate utilization test.
• The lower case "i" is for "in" as the Citrate test requires coliform pure isolate samples to be placed "in Citrate agar".

General Microbiology Short Review Guide

Microbiology Study Guide for Media, Tests and Specimen Processing

Fungal Structures and Mycology Terminologies Essential for Identification

Clinical mycology remains more of a descriptive art than an analytical science. You may find that the identification of fungi requires a greater development of your visual acuity than was necessary in bacteriology. There are also fewer biochemical tests available to aid in the differential identification of fungi. As a result you will spend considerable time in the laboratory visually examining fungal cultures in slide culture preparations. You will identify characteristic fungal structures by observing colonial growth both macroscopically and microscopically.

A thorough understanding of correct fungal terminology is of critical importance. You may find that the terms used to describe fungi are unusual, at times redundant, and often very confusing. We have attempted to simplify the jumbled jargon by providing you with the following list of terms that will be used most often in the laboratory section of this course. Although most of these terms have already been introduced in the lecture notes, they have been included in the laboratory manual as well.

Riddell Method- Slide Culture Preparation for Fungal Identification

In order to accurately identify many fungi specifically molds, it is essential to observe the precise arrangement of the conidiophores and the way in which spores are produced. Riddell’s simple method of slide culturing permits fungi to be studied virtually in situ with as little disturbance as possible because many fungi form very fragile reproductive structures which are at least partially disrupted by even the most careful manipulation.

Agars To Be Used

One plate of nutrient agar; potato dextrose is recommended, however, some fastidious fungi may require harsher media to induce sporulation like Cornmeal agar or Czapek Dox agar. Sabourauds Destrose Agar (SAB) may also be used.

Sexually Transmitted Disease (STD) Facts, Complications and Prevention

Sexually Transmitted Disease (STD) Facts

• Sexually transmitted infections (STIs) are mostly spread from one infected person to another through sexual intercourse. Some infections may also be transmitted from mother to child during pregnancy and childbirth. Another way that infections are passed on is through the sharing of blood products or tissue transfers. Some diseases caused by STIs include syphilis, AIDS and cervical cancer.

• STIs often exist without symptoms, particularly in women. Thus, men and women with sexual partners who have STI symptoms should seek care regardless of a lack of signs. Whenever an infection is diagnosed or suspected, effective treatment should be provided promptly to avoid complications.

• STIs disproportionately affect women and adolescent girls. Every year, one in 20 adolescent girls gets a bacterial infection through sexual contact, and the age at which infections are acquired is becoming younger and younger. Improving awareness and knowledge of STIs and how to prevent them among adolescents should be part of all sexual health education and services.

Acetate Utilization Test: Method, Principle and Interpretation

Principle:

This test is used to determine if an organism can use acetate as the sole source of carbon. If so, breakdown of the sodium acetate causes the pH of the medium to shift toward the alkaline range, turning the indicator from green to blue.

Method:

1. With a straight inoculating needle, inoculate acetate slant lightly from an 18- 24 hour culture. Do not inoculate from a broth culture, because the growth will be too heavy.

2. Incubate at 35 degree Celsius for up to 7 days.

Human Papillomavirus (HPV): Facts and Vaccines

Facts:

• HPV vaccines are close to 100% effective in preventing HPV infection and precancerous conditions that may lead to cervical cancer, if the vaccine is given before a person is exposed to the HPV types contained in the vaccine.
• HPV vaccines appear to be safe and generally are well tolerated.
• Two HPV vaccines have been developed. Both prevent infection with type 16 and -18, and may prevent up to 70% of cervical cancers. One of the vaccines also prevents infection with HPV-6 and -11, and may prevent about 95% of genital warts.

Acetamide Utilization Test: Principle, Procedure and Interpretation

Principle:

This test is used to determine the ability of an organism to use acetamide as the sole source of carbon. Bacteria that can grow on this medium deaminate acetamide to release ammonia. The production of ammonia results in a pH- driven color change of the medium from green to royal blue.

Method:

1. Inoculate acetamide slant lightly with a needle using growth from an 18-24 hour culture.
2. Do not inoculate from a broth culture, because the growth will be too heavy.

Types of Microorganisms: Description and Morphology

Microorganisms, also known as "microbes", are single cell or multicellular organisms that are mostly microscopic in nature but there are some which are macroscopic and visible to the naked eye. Microorganisms are very diverse and they include the following:

Bacteria

Bacteria

Bacteria (singular: bacterium) are relatively simple, single-celled (unicellular) organisms. Because their genetic material is not enclosed in a special nuclear membrane, bacterial cells are called prokaryotes, from Greek words meaning prenucleus. Prokaryotes include both bacteria and archaea.

Bacterial cells generally appear in one of several shapes. Bacillus (rodlike), (spherical or ovoid), and spiral (corkscrew or curved) are among the most common shapes, but some bacteria are star shaped or square. Individual bacteria may form pairs, chains, clusters, or other groupings; such formations are usually characteristic of a particular genus or species of bacteria.

Coagulase Test: Slide and Tube Method

Principle:

This test is used to differentiate Staphylococcus aureus (positive) from coagulase- negative staphylococci (negative). S. aureus produces two forms of coagulase: bound and free. Bound coagulase, or “clumping factor”, is bound to the bacterial cell wall and reacts directly with fibrinogen. This results in an alteration of fibrinogen so that it precipitates on the staphylococcal cell, causing the cells to clump when a bacterial suspension is mixed with plasma. The presence of bound coagulase correlates well with free coagulase, an extracellular protein enzyme that causes the formation of clot when S. aureus colonies are incubated with plasma. The clotting mechanism involves activation of a plasma coagulase- reacting factor (CRF), which is modified or derived thrombin molecule, to form a coagulase- CRF complex. This complex in turn reacts with fibrinogen to produce the fibrin clot.

Dengue Facts: Transmission, Treatment and Prevention

Key Facts About Dengue

• Dengue is a mosquito-borne viral infection.
• The infection causes flu-like illness, and occasionally develops into a potentially lethal complication called severe dengue.
• The global incidence of dengue has grown dramatically in recent decades.
• About half of the world's population is now at risk.
• Dengue is found in tropical and sub-tropical climates worldwide, mostly in urban and semi-urban areas.
• Severe dengue is a leading cause of serious illness and death among children in some Asian and Latin American countries.