Direct Epifluorescence Microscopy Research Articles

Effect of milk proteins on adhesion of bacteria to stainless steel surfaces.

Stainless steel coupons were treated with skim milk and subsequently challenged with individual bacterial suspensions of Staphylococcus aureus, Pseudomonas fragi, Escherichia coli, Listeria monocytogenes, and Serratia marcescens. The numbers of attached bacteria were determined by direct epifluorescence microscopy and compared with the attachment levels on clean stainless steel with two different surface finishes. Skim milk was found to reduce adhesion of S. aureus, L. monocytogenes, and S. marcescens. P. fragi and E. coli attached in very small numbers to the clear surfaces, making the effect of any adsorbed protein layer difficult to assess. Individual milk proteins alpha-casein, beta-casein, kappa-casein, and alpha-lactalbumin were also found to reduce the adhesion of S. aureus and L. monocytogenes. The adhesion of bacteria to samples treated with milk dilutions up to 0.001% was investigated. X-ray photoelectron spectroscopy was used to determine the proportion of nitrogen in the adsorbed films. Attached bacterial numbers were inversely related to the relative atomic percentage of nitrogen on the surface. A comparison of two types of stainless steel surface, a 2B and a no. 8 mirror finish, indicated that the difference in these levels of surface roughness did not greatly affect bacterial attachment, and reduction in adhesion to a milk-treated surface was still observed. Cross-linking of adsorbed proteins partially reversed the inhibition of bacterial attachment, indicating that protein chain mobility and steric exclusion may be important in this phenomenon.

Effectiveness of cleaning techniques used in the food industry in terms of the removal of bacterial biofilms.

The effectiveness of cleaning was investigated through food factory trials and laboratory experiments using a naturally occurring biofilm from a food factory environment and generated biofilms. The efficacy of factory cleaning and disinfection programmes was assessed by swabbing and total viable count (TVC) analysis of surfaces before cleaning, after cleaning and after disinfection. Cleaning produced a 0.91 log reduction in the attached population. Investigation of the effectiveness of a variety of cleaning methods in the removal of a naturally occurring food factory biofilm showed that the high pressure spray and the mechanical floor scrubber, which use a high degree of mechanical action, were most effective. Cleaning trials with biofilms of Pseudomonas aeruginosa or Staphylococcus aureus showed that spraying with water at pressures of 34.5, 51.7 and 68.9 bar did not significantly increase the removal, as assessed by direct epifluorescent microscopy (DEM) and swabbing and TVC analysis, beyond the three log reduction observed at 17.2 bar. The effect of spray time at 17.2 bar showed that increasing spray time from 1 to 10 s did not significantly increase removal of Ps. aeruginosa biofilm. Investigation of the optimum distance of the spray lance from the surface at 17.2 bar was found to be between 125 and 250 mm. The use of an alkaline, acidic or neutral detergent prior to spraying with water at 17.2 bar did not significantly increase the removal of Ps. aeruginosa or Staph. aureus. However, the acidic and alkaline products significantly (P = 0.05) affected the viability of Staph. aureus and Ps. aeruginosa, respectively, thereby minimizing the potential for the spread of contamination.

Bacillus spores and moulding with TTC agar: A useful method for the assessment of food processing equipment cleanability

In the present work a powerful and practical method for the assessment of food processing equipment clean‐ability is described. Surfaces were soiled with Bacillus spores. After cleaning, surfaces were overlaid with agar containing a tetrazolium salt (TTC) which stained the growing colonies red. Stained colonies could be readily detected and counted. The poorer hygienically designed areas were easily observed and contamination levels could be defined. The TTC method was compared to other classical techniques such as contact plates, direct epifluorescence microscopy (DEM) and Shapton and Hindes (SH) agar. Recovery of bacteria using the TTC method was found to be a good complement to DEM and more accurate than contact plates and SH agar. Cleanability tests on closed equipment are also reported showing the utility of the TTC method.

Frequency distribution of Candida albicans blastospores adhered to mucosal epithelial cells in-vitro.

Although several methods are available for examination of microbial adherence to epithelial cells, these do not distinguish between adherence of viable and non-viable micro-organisms. This study reports the use of acridine orange-stained blastospores of Candida albicans in conjunction with direct epifluorescence microscopy to determine viable (orange-fluorescing) and non-viable (green-fluorescing) blastospore adherence to buccal epithelial cells. The method was also employed to examine the effects of chlorhexidine treatment at subminimum inhibitory concentrations on the adherence of viable and non-viable blastospores. There was good correlation in the assessment of blastospore viability between the direct epifluorescence microscopy technique and the standard serial dilution and plating method for viable counting, confirming the reliability of direct epifluorescence microscopy. Chlorhexidine treatment before acridine orange staining did not alter this assessment of viability. Blastospore adherence to buccal epithelial cells resulted in a similarly skewed distribution whether examined using a crystal violet stain in conjunction with light microscopy or using direct epifluorescence microscopy, therefore validating the direct epifluorescence microscopy technique for the enumeration of blastospore adherence. Chlorhexidine treatment (0.0005% v/v, 30 min) of either blastospores or buccal epithelial cells altered the distribution of adherent blastospores per epithelial cell by increasing the number of epithelial cells having no adherent blastospores. No differences in adherence were, however, observed between blastospore or epithelial cells after treatment with this agent. Examination of the adherence of viable and non-viable blastospores to buccal epithelial cells using direct epifluorescence microscopy revealed a greater adherence capacity of non-viable than viable blastospores for buccal epithelial cells. Treatment of blastospores with chlorhexidine altered the frequency distributions of viable and non-viable blastospores with lower numbers of blastospores adherent per epithelial cell. The larger reduction in adherent viable blastospores in comparison with their non-viable counterparts is, however, an important observation which might have clinical relevance. Microbial cells adhere to epithelial cells resulting in a skewed distribution; study of this distribution gives useful information about the adherence process. Viable and non-viable components of a microbial population have different adherence capabilities and treatment of such populations with an antimicrobial agent exerting anti-adherent activity at sub-minimum inhibitory concentrations reduces the amount of adherence of these viable/non-viable components to different extents.

Towards a Non‐Terminal Viability Assay for Foraminiferan Protists

ABSTRACT. Epifluorescence microscopy and spectrofluorimetry were investigated as possible non‐terminal methods to distinguish live from dead foraminifera. Seven fluorogenic probes (diacetates of fluorescein [FDA], carboxyfluorescein, dichlorofluorescein, and carboxyeosin; AM‐esters of biscarboxyethylcarboxyfluorescein [BCECF‐AM], calcein, and calcein blue) were tested on Allogromia laticollaris. The probes that consistently produced the brightest fluorescence signals (BCECF‐AM and FDA) were judged non‐toxic to Allogromia, on the basis of short‐term pseudopodial deployment and long‐term reproduction assays. Once protocols were established, these two probes were tested on 13 additional benthic foraminiferal species. We found that BCECF‐AM is the most suitable probe for direct epifluorescence microscopy of metabolically active foraminifera, especially tectinous and transparent calcareous species. Using spectrofluorimetry, FDA showed promise for opaque species because fluorescence is detected in the incubation media after its release from the cell. However, both approaches could only be used with confidence in light of appropriate controls established for each species examined.

Routine monitoring of Cryptosporidium oocysts in water using flow cytometry

A flow cytometric method for the routine analysis of environmental water samples for the presence of Cryptosporidium oocysts has been developed. It uses a Coulter Epics Elite flow cytometer to examine water samples and to separate oocysts from contaminating debris by cell sorting. The sorted particles are then rapidly screened by microscopy. The method has been evaluated and compared with direct epifluorescence microscopy on 325 river, reservoir and drinking water samples. The technique was found to be more sensitive, faster and easier to perform than conventional epifluorescent microscopy for the routine examination of water samples for Cryptosporidium.

The effect of some environmental parameters on surface colonization by microorganisms

The occurrence of biofouling in water systems results from microbial growth on submerged surfaces. Biofouling-related problems are often overcome by chemical treatment. However, situations often develop which necessitate a complete shutdown in order to allow mechanical cleaning of a system as well as replacement of corroded piping material. Strategies additional to chemical treatment are therefore needed to control biofouling. Such strategies may include physical or chemical alterations to a water system to create conditions less favourable for microbial activity. Different fluid velocities were simulated in a single continuous flow reactor which enabled the comparison of bacterial colonization of surfaces at six different fluid velocities while all other parameters were kept constant. As expected, the highest colonization rates occurred at conditions of zero flow, with a decrease in fouling rates with an increase of fluid velocity. In a second set of experiments, the influence of dissolved organic carbon concentration on biofouling of different types of surfaces was studied. In these experiments, highest colonization rates occurred on stainless steel, followed by aluminium and glass. Direct epifluorescent microscopy techniques were employed to enumerate sessile bacteria. There was a progressive increase in corrosion rate with increases in sulphate concentration up to sulphate levels greater than 600 mg/l after which there was no further marked increase in corrosion rate.

The use of epifluorescence microscopy to determine surface hygiene

Within the food industry a need has been established for rapid techniques to assess the microbial contamination on food contact surfaces. A method was developed, by adapting the DEFT technique, to stain and enumerate microorganisms by direct epifluorescent microscopy. The method was evaluated by developing biofilms of various organism on a range of food grade surfaces, and in factory environments using test sampling plates. Cleaning trials were undertaken to examine the ability of the method to detect low levels of surface contamination. The method was shown to enumerate surface organisms rapidly (⩽15 min) within the range 3 × 10 3−5 × 10 7 colonies/cm 2. No difficulties were experienced in evaluating different organisms nor the majority of food grade surfaces. The technique may prove useful in allowing a real-time control for the assessment of surface hygiene.

The use of direct epifluorescent microscopy (DEM) and the direct epifluorescent filter technique (DEFT) to assess microbial populations on food contact surfaces.

Two rapid methods, direct epifluorescent microscopy (DEM) and the direct epifluorescent filter technique (DEFT) on swab resuspension fluids, were compared with the traditional total viable count (TVC) on swab resuspension fluids for their ability to enumerate surface populations of attached bacteria. The degree of error in estimating surface populations was shown to be significantly less with DEM than DEFT followed by TVC. DEM estimated populations in the range 3 x 10(3) to 5 x 10(7) colonies/cm2 whilst DEFT enumerated populations above 3 x 10(4) colonies/cm2 and TVC above 3 x 10(5) colonies/cm2 (as measured by DEM). Swabbing was shown to remove a constant proportion of organisms from the surface populations tested, although below 3 x 10(5) colonies/cm2 most of the organisms remained in the cotton matrix and were difficult to resuspend. DEFT was more able to enumerate swab resuspension fluids obtained from surface populations below 3 x 10(5) colonies/cm2 than was TVC.

Relations entre les compositions en acides amines des particules en voie de sedimentation, du contenu intestinal des holothuries abyssales et du sediment environnant

The amino acid composition of the gut content of two species of deposit-feeding holothurians ( Deima validum validum and Pseudostichopus villosus) is compared with that of the sediment trap particles and the surrounding sediment collected from the Demerara abyssal plain in the equatorial Atlantic Ocean at a depth of 4800 m. Amino acid concentrations are ∼ 10, ∼ 50 and ∼ 100 μmol g −1 in superficial sediment, sediment trap particles and ingested sediments, respectively. Compositions of the latter two are nearly identical and differ slightly from that of the former. No selective absorption of amino acids is noticed through the digestive tract. The acid-soluble (cold 2 N HCl) hydrolysed category, nearly absent in sediment trap particles from that depth as well as in superficial sediment, increases in gut contents for both species as a result of digestive system action on particulate matter. Glutamic acid which is largely predominant in this fraction may be partly secreted by the organisms or may result from bacterial activity. Similarly diaminopimelic acid (DAP) is important in gut contents only, but entirely in the free form. The bacterial cell numbers calculated from DAP concentrations are 100-fold that of direct epifluorescent microscopy counts but cannot be considered as the numbers of visible cells because the cell-wall mureid complex containing DAP is normally insoluble in water. Nevertheless, DAP concentrations lead pointedly to the fact that remains of larger bacterial populations or more intense bacterial activity exist in gut content compared to that of sinking particles and superficial sediment. In the superficial sediment, β-alanine and γ-aminobutyric acid account for about 8 mol%, almost entirely in the residual hydrolysed category. In sinking particles and gut contents they account for 1.5 mol% only, but their total amounts are similar to that of the superficial sediment. In gut contents about 40% of β-alanine and γ-aminobutyric acid are found in acid- and alkali-soluble hydrolysed categories, maybe as a result of the general decomposition of ingested organic matter. Absolute concentrations do not appear to change through the digestive tract. The inefficiency of organism assimilation systems and the intimate contact between mineral particles and digested organic compounds which occur in the digestive tract of deposit-feeders may be partly responsible for their increased relative abundance in pelagic sediments.