Effect of heat-staining procedure on the Gram staining properties of mycobacteria.

The microacoustic methods of biomedical analysis, implemented on piezoelectric crystals and ceramics, are becoming increasingly popular due to the fact of their potential for integration into laboratories-on-a-chip, biochips, and biosensors as functional elements of biosensors. An important stage in diagnostics of infectious diseases is the identification of pathogens. One possible applications of such a sensor is an alternative to the time- and labor-consuming Gram method of discriminating bacteria according to the composition of their cell walls. Thus, bacteria, which in a Gram staining procedure do not decolor after application of the dye solution, are classified as Gram-positive (G(+)). They are surrounded with a thick peptidoglycan layer that is pulpy and dampens acoustic waves. While Gram-negative (G(–)) bacteria, which acquire a red color in a Gram procedure, are covered with a thin and springy layer, demonstrating resonance effects when interacting with acoustic fields. Thus, G(+) and G(–), which are differently colored in Gram procedures, also react differently to an external acoustic field: for G(–) bacteria, this was a sharp decrease in the Q-factor of the “resonator–suspension” system and a shift of the resonance curve to lower frequencies. While for G(+) bacteria, although a certain shift of the resonance curve was also observed, the bandwidth of the resonance curve practically did not change. This effect was studied for L. acidophilus (G(+)) and Escherichia coli (G(–)) bacilli with quarts resonators of 4 MHz, 5 MHz, and 10 MHz. The biosensor was tested using Lactobacillus fermentum, E. coli M-17, Bifidobacterium bifidum, Burkholderia cepacian, and Staphylococcus aureus. At this stage, it has been demonstrated that the method is particularly effective for discriminating bacteria of a similar shape, such as, for example, cocci. The discrimination of the Gram factor for cocci and bacilli was less accurate and needs further studies for selection of precise resonance frequencies.

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Gram Staining: a Comparison of Two Automated Systems and Manual Staining

Journal of Clinical Microbiology ◽

10.1128/jcm.01914-20 ◽

2020 ◽

Vol 58 (12) ◽

Author(s):

Neele J. Froböse ◽

Sara Bjedov ◽

Franziska Schuler ◽

Barbara C. Kahl ◽

Stefanie Kampmeier ◽

...

Keyword(s):

Public Service ◽

The United States ◽

University Hospital ◽

Clinical Samples ◽

Staining Procedure ◽

Automated Systems ◽

The Public ◽

Gram Staining ◽

Hands On ◽

The Mean

ABSTRACT Various Gram staining automated systems are available to accelerate and standardize the staining process, but a systematic comparison of different systems is largely lacking. The objective of this study was to evaluate two devices in comparison to manual Gram staining. Clinical samples (n = 500; University Hospital Münster, Germany; May to June 2020) were simultaneously Gram stained manually and with two automated Gram stainers (Previ Color Gram, bioMérieux, and ColorAX2, Axonlab). The quality was assessed based on four criteria: (i) homogeneous staining of bacteria/fungi, (ii) uniform staining of the background, (iii) absence of staining artifacts, and (iv) congruency between culture and microscopy. Each criterion was rated with 0 (absence) or 1 (presence) point to calculate a quality score (0 to 4 points). The costs for each staining procedure were calculated based on consumables and hands-on time (applying the average wage of a laboratory technician in the public service for Germany and the United States). The mean (± standard deviation [SD]) quality scores were comparable for manual staining (3.06 ± 0.91) and Previ Color Gram (3.04 ± 0.90; P = 0.6), while significantly lower scores were achieved by ColorAX2 (2.57 ± 1.09; P < 0.0001). The total cost per Gram stain was €1.13/$1.34 for Previ Color Gram, €0.80/$0.83 for manual, and €0.60/$0.71 for ColorAX2, respectively. The quality and costs per slide vary significantly between instruments of different manufacturers.

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Assessment of the Coliform Bacterial Load of Some Drinking Water Sources in Dutse Metropolis of Jigawa State Nigeria

Scientific Review ◽

10.32861/sr.66.48.52 ◽

2020 ◽

pp. 48-52

Author(s):

Ibrahim Khalil Abubakar ◽

Mustapha Mohammed Abubakar ◽

Muhammad M. Abubakar

Keyword(s):

Drinking Water ◽

Membrane Filtration ◽

Bacterial Load ◽

Staining Procedure ◽

Faecal Coliforms ◽

Biochemical Tests ◽

E Coli ◽

Gram Staining ◽

Water Companies ◽

Sachet Water

Drinking water samples from 5 sachet water companies, 3 boreholes and 2 taps, collected from different locations of Dutse Metropolis of Jigawa State, Nigeria were analysed for coliform bacterial counts using the Membrane Filtration Technique. All the samples contained some amounts of total coliforms, but mostly within permissible levels. Thirty three percent (33%) of the samples from borehole, 60% from sachet water and 100% from the taps contained faecal coliforms, which indicates contamination. Cultures of the faecal coliforms obtained were morphologically identified using the gram-staining procedure and some series of biochemical tests were carried out in order to identify the organisms. The identified organisms were Escherichia coli (E. coli), Klebsiella sp. and Citrobacter sp. Presence of coliforms above the regulatory set standards indicates contamination and un-safeness of the water for drinking. Presence of organisms such as E. coli, Klebsiella sp. and Citrobacter sp. necessitates improvement in monitoring and water hygiene practices to improve the quality of drinking water in the study area.

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Evaluation of an Automatic Gram-Staining Machine

Journal of Milk and Food Technology ◽

10.4315/0022-2747-38.5.262 ◽

1975 ◽

Vol 38 (5) ◽

pp. 262-263

Author(s):

DANIEL Y. C. FUNG

Keyword(s):

Conventional Method ◽

Microscopic Examination ◽

Efficiency Factor ◽

Staining Procedure ◽

Bacterial Cultures ◽

Gram Staining ◽

Overall Efficiency

An automated staining machine—the Microstainer II—was evaluated for its effectiveness in the Gram staining of bacterial cultures. The conventional hand staining procedure was used as a comparison. Microscopic examination of stained slides revealed that both techniques gave correct Gram reaction of the bacterial cultures tested. The Microstainer II has an overall “efficiency factor” of 10 × compared to the conventional method.

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Ca-Histochemistry in slime mold plasmodia

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081516 ◽

1978 ◽

Vol 36 (2) ◽

pp. 130-131

Author(s):

Ulrich Dierkes

Keyword(s):

Physarum Polycephalum ◽

Carbon Coating ◽

Freeze Drying ◽

Freeze Fracture ◽

Freeze Substitution ◽

Uranyl Acetate ◽

Slime Mold ◽

Staining Procedure ◽

Protoplasmic Streaming ◽

Intracellular Ca

Calcium is supposed to play an important role in the control of protoplasmic streaming in slime mold plasmodia. The motive force for protoplasmic streaming is generated by the interaction of actin and myosin. This contraction is supposed to be controlled by intracellular Ca-fluxes similar to the triggering system in skeleton muscle. The histochemical localisation of calcium however is problematic because of the possible diffusion artifacts especially in aquous media.To evaluate this problem calcium localisation was studied in small pieces of shock frozen (liquid propane at -189°C) plasmodial strands of Physarum polycephalum, which were further processed with 3 different methods: 1) freeze substitution in ethanol at -75°C, staining in 100% ethanol with 1% uranyl acetate, and embedding in styrene-methacrylate. For comparison the staining procedure was omitted in some preparations. 2)Freeze drying at about -95°C, followed by immersion with 100% ethanol containing 1% uranyl acetate, and embedding. 3) Freeze fracture, carbon coating and SEM investigation at temperatures below -100° C.

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Dual stain kit for the microscopic gram classification of ocular infection bacteria

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100147089 ◽

1993 ◽

Vol 51 ◽

pp. 248-249

Author(s):

Jacob S. Hanker ◽

Dale N. Holdren ◽

Kenneth L. Cohen ◽

Beverly L. Giammara

Keyword(s):

Contact Lenses ◽

Ocular Infection ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Gram Staining ◽

Ocular Infections ◽

Different Types ◽

Culture Positive ◽

Increased Susceptibility

Keratitis and conjunctivitis (infections of the cornea or conjunctiva) are ocular infections caused by various bacteria, fungi, viruses or parasites; bacteria, however, are usually prominent. Systemic conditions such as alcoholism, diabetes, debilitating disease, AIDS and immunosuppressive therapy can lead to increased susceptibility but trauma and contact lens use are very important factors. Gram-negative bacteria are most frequently cultured in these situations and Pseudomonas aeruginosa is most usually isolated from culture-positive ulcers of patients using contact lenses. Smears for staining can be obtained with a special swab or spatula and Gram staining frequently guides choice of a therapeutic rinse prior to the report of the culture results upon which specific antibiotic therapy is based. In some cases staining of the direct smear may be diagnostic in situations where the culture will not grow. In these cases different types of stains occasionally assist in guiding therapy.

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A pre-embedding double staining procedure used to observe the effect of fixation on the activity of intercellular adhesion molecule on T and B cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100159436 ◽

1990 ◽

Vol 48 (3) ◽

pp. 378-379

Author(s):

Jane E. Ramberg ◽

Shigeto Tohma ◽

Peter E. Lipsky

Keyword(s):

B Cells ◽

Adhesion Molecule ◽

Colloidal Gold ◽

Intercellular Adhesion ◽

Intercellular Adhesion Molecule ◽

Staining Procedure ◽

Double Staining ◽

T And B Cells ◽

Microtiter Plates ◽

Streptavidin Peroxidase

Intercellular adhesion molecule (ICAM-1) appears to be a ligand for LFA-1 dependent adhesion in T cell mediated cytotoxcity. It is found on cells of both hematopoietic and non-hematopoietic origin. While observing the activity of ICAM-1 on the surfaces of interacting T and B cells, we found that we could successfully carry out a pre-embedding double staining procedure utilizing both colloidal gold and peroxidase conjugated reagents.On 24-well microtiter plates, mitomycin-treated T4 cells were stimulated with 64.1 (anti-CD3) for one hour before the addition, in some instances, of B cells. Following a 12-48 hour incubation at 38°C, the cells were washed and then immunostained with a colloidal gold conjugated RFB-4 (anti-CD22); biotinylated R6.5 (anti-ICAM-1); followed by streptavidin/peroxidase. This method allowed us to observe two different antigens without concern about possible cross-reaction of reagents. Because we suspected ICAM-1 and R6.5 were sensitive to fixation, we tried varying concentrations of fresh paraformaldehyde before R6.5, after R6.5 and after streptavidin/peroxidase. All immunostaining and washing was done on ice with ice cold reagents.

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