Occurrence and distribution of sucrose-metabolizing enzymes in oral streptococci.

Specific growth rates, growth yields, and the level and cellular distribution of three sucrose-metabolizing enzyme activities were determined for seven oral streptococci (Streptococcus mutans strains E49, BHT, 10449, SL-1, and LM-7, S. sanguis 10558, and S. salivarius 25975). Cultures were grown in a fermentor at pH 6 with either 20 mM glucose or 10 mM sucrose.Generation times varied between 21 and 70 min. Whereas some strains grew 10 to 50% more slowly with sucrose than with glucose, others did not. Growth was always logarithmic, and the growth yields were similar. Glcosyl transferase (EC 2.4.1.5) was largely extracellular; in sucrose cultures it was appreciably lower, but no major shift to a cell-associated form was found. In glucose cultures, the activity varied between 4 and 140 IU per 6-liter culture. The glucan formed was mostly or exclusively water insoluble. Glcosyl transferase was stimulated weakly (60% or less) by various dextrans. Fructosyl transferase (EC 2.4.1.10) was primarily extracellular (except in glucose cultures of S. salivarius) and varied between 0 and 337 IU/culture. In S. salivarius, the extracellular fructosyl transferase was induced by sucrose. In all S. Mutans cultures, the total fructosyl transferase activity was lower after growth with sucrose. All strains had extra- and intracellular invertase (EC 3.2.1.26) activity. Total levels varied between 210 and 3,500 IU/culture. Less extracellular activity was present in sucrose cultures. Only S. salivarius had appreciable activity in the cellular particulate fraction. Invertase activity was significantly higher than the combined glucosyl and fructosyl transferase activities in all cultures.     

Analysis of growth rate in sucrose-supplemented cultures of Streptococcus mutans.

In the presence of sucrose, Streptococcus mutans grows in large glucan-containing aggregates. Because of reports of linear rather than exponential growth of sucrose-grown cultures, the kinetics of growth of sucrose-grown cultures of S. mutans strain OMZ-176 were compared with those of glucose-grown cultures. Culture turbidity measurements indicated that growth of sucrose cultures was slower, did not follow exponential kinetics, and slowed and stopped at lower absorbance values than did glucose-grown cultures. However, measurements of the rates of accumulation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein using fully equilibrated radioactively labeled precursors of each of these macromolecular species in sucrose and glucose-grown cultures showed that: (i) for glucose cultures the synthesis of each of the three informational molecules occurred at the same exponential rate, which was identical to the rate of turbidity increase; (ii) for sucrose cultures each macromolecular species was synthesized at the same exponential rate and these rates were identical to the rate of increase of turbidity of the glucose-grown culture for periods of up to 7 h. Furthermore, the ratios of DNA to RNA, RNA to protein, and protein to DNA for the sucrose cultures were identical to those for the glucose cultures for up to 10 doublings. From these data it was concluded that in the presence of sucrose S. mutans grows in a balanced fashion at the same exponential rate as it does in glucose. The deviation from an exponential growth model of the absorbance in sucrose cultures was attributed to an optical artifact due to the formation of large glucan-containing aggregates of cells. The addition of dextranase to sucrose cultures resulted in cultures which increased in turbidity at the same exponential rate as glucose-grown cultures, without affecting the rate or extent of macromolecular synthesis.     

Production of Extracellular and Cell-Associated Glucosyltransferase Activity by Streptococcus mutans During Growth on Various Carbon Sources

The production of extracellular and cell-associated glucosyltransferase activity by Streptococcus mutans strain GS-5 was examined during growth on various carbon sources in a chemically defined medium. S. mutans cells produced glucosyltransferase activity only during logarithmic growth when glucose, fructose, mannitol, or sorbitol was the sole carbon source. Cells growing on mannitol or sorbitol produced approximately half as much extracellular glucosyltransferase activity as cells growing on glucose, although the proportions of the glucosyltransferase activity capable of synthesizing insoluble glucans were similar. Cells growing on fructose produced slightly more extracellular glucosyltransferase activity than cells grown on glucose, yet the proportion of the glucosyltransferase activity capable of synthesizing insoluble glucans was again similar to glucose cultures. S. mutans cells growing in the presence of both glucose and mannitol displayed diauxic growth and initial preferential utilization of glucose. Glucosyltransferase enzyme production occurred only during the phases of cell growth in the presence of the two carbon sources. The cell-associated glucosyltransferase activities of glucose-, fructose-, mannitol-, and sorbitol-grown cells were relatively low, yet all the cells were capable of adherence to glass in the presence of sucrose. When glucose-containing cultures of S. mutans were supplemented with sucrose, extracellular glucosyltransferase activity first became cell associated and then appeared to become inactivated, presumably due to the accumulation of insoluble glucans.     

Analysis of baculovirus aggregates using flow cytometry

Aggregation of viral particles represents a significant problem for baculoviral stock processing and storage. Aggregation may also affect the results of viral particle counting. A method using flow cytometry was previously developed in our lab to measure the concentration of baculovirus particles produced in insect cell cultures. In the present study, the use of the flow cytometry method was extended to the detection of baculovirus aggregates. Flow cytometry analysis of freshly prepared baculovirus stocks, stained with SYBR Green, generally exhibited a single unimodal distribution; while, baculovirus stocks stored at 4 degrees C for a few months exhibited a bimodal distribution of the fluorescent intensity signal. The bimodal distribution was associated with a decrease in the size of the original viral population and an emergence of a new viral population with a high fluorescence intensity. Treatment of these samples with an endonuclease (Benzonase) confirmed that the new population observed in the flow cytometry analysis is not free cellular DNA. Filtration through 0.22 and 0.45 microm membranes of the stored samples prior to flow cytometry analysis confirmed that the high fluorescence intensity population involved particles larger than a single baculovirus. Exposing freshly amplified baculovirus stocks with a unimodal distribution to a pH of 5.3, a condition known to induce aggregation, showed the emergence of a second population with a bimodal distribution. These results suggest that flow cytometry analysis could be used to detect baculovirus aggregates. The aggregates were associated with high fluorescence intensity populations and the mean green fluorescence intensity of these populations could be used as an indicator of the mean aggregate size.     

Effect of growth conditions on sucrose phosphotransferase activity of Streptococcus mutans.

Sucrose and glucose phosphoenolpyruvate-dependent phosphotransferase (PTS) activities were studied in growing cultures of Streptococcus mutans serotype c and d/g cells adapted to either glucose or sucrose. Both acid production and optical absorbance were used to monitor growth in pH-controlled defined growth medium. The sucrose PTS activity appeared to be significant only under conditions of substrate limitation or slow growth as a result of low environmental pH. However, under environmental conditions which permitted rapid growth sucrose PTS activity appeared to be repressed, and only when the cells approached substrate-limited stationary phase after growth on high sucrose-supplemented medium was significant sucrose PTS activity again observed. A mutant apparently defective in sucrose PTS activity grew rapidly and produced acid under conditions of high environmental sucrose level but showed no sucrose PTS activity when the culture approached stationary phase. The mutant, however, after adaptation to glucose, demonstrated significant glucose PTS once the culture had attained the stationary growth phase. During diauxie growth in the presence of glucose and sucrose, there were sequential apparent inductions and repressions of glucose and sucrose PTS activities corresponding to decreases and increases of growth rate on the two substrates. Thus, S. mutans possesses at least two transport mechanisms for each substrate studied. One system (PTS) functions under conditions permitting slow growth and another functions under conditions permitting rapid growth.     

Carbon source regulation of beta-galactosidase biosynthesis in Penicillium chrysogenum.

Growth and beta-galactosidase activity of the penicillin producer industrial Penicillium chrysogenum NCAIM 00237 strain were examined using different carbon sources. Good growth was observed using glucose, sucrose, glycerol and galactose, while growth on lactose was substantially slower. beta-Galactosidase activity was high on lactose and very low on all the other carbon sources tested. In glucose grown cultures after exhaustion of glucose as repressing carbon source a derepressed low level of the enzyme was observed. cAMP concentration in lactose grown cultures was relatively high, in glucose grown cultures was low. Caffeine substantially decreased glucose consumption and growth but did not increase beta-galactosidase activity and did not prevent glucose repression which rules out the involvement of cAMP in the regulation of beta-galactosidase biosynthesis in Penicillium chrysogenum.     

Effect of carbohydrate source and growth conditions on the production of lipoteichoic acid by Streptococcus mutans Ingbritt.

Streptococcus mutans Ingbritt was grown in a chemostat at defined dilution rates and pH values and under carbohydrate limitation. At a constant dilution rate of D = 0.1 h-1 and with either 0.5% glucose or 0.5% sucrose, the amounts of both cellular and extracellular lipoteichoic acid increased as the culture pH increased from 5.0 to 7.5. At a constant pH of 6.0, the amount of cellular lipoteichoic acid formed by cultures growing in 0.2% or 0.5% glucose was relatively constant over a range of dilution rates, although the amount of extracellular lipoteichoic acid formed in 0.2% glucose at intermediate dilution rates was less than that formed in 0.5% glucose. Organisms grown in 0.5% sucrose at pH 6.0 contained increasing amounts of cellular lipoteichoic acid as the dilution rate was increased. A comparison of the amounts of cellular lipoteichoic acid formed by organisms growing at D = 0.5 h-1 and pH 6.0 in glucose, sucrose, fructose, or mixtures of glucose and fructose in limiting amounts suggested that the enhanced production of lipoteichoic acic by sucrose-grown organisms was due to the fructose component. The culture fluids from both glucose- and sucrose-grown organisms contained detectable amounts of serotype c antigen, whereas glucose-grown cultures also contained significant amounts of an extracellular hexose-containing polymer.     

Quantitation and phenotyping of T cell clones by flow cytometry.

In order to efficiently analyze large numbers of T cell clones, a method for analysis of small cell cultures by flow cytometry was developed. The aim was to assess cloning efficiency, growth rate and phenotype of T cell clones. The reliability of the flow cytometer for quantitation of cell populations was documented by repeated analysis of manually counted cell samples. In the concentration range from 3000 to 195,000 cells/ml, the correlation coefficient between counts obtained by the flow cytometer and manual cell counts was 0.999 and within-assay coefficients of variation were below 4%. Cell cultures containing less than 200 cells were reliably quantified. The technique was applied for analysis of T cell clonings with different mitogens and in the presence of varying amounts of serum. To reduce time and labour, the cultures were analyzed only 10 days after cloning, when the clones contained less than 100,000 cells. The sensitivity of the flow cytometer in the detection of immunolabeled cells made further expansion of cell cultures unnecessary, thus greatly reducing manual labour and experiment turnover time. The commonly used mitogens phytohemagglutinin (PHA) and the antibody OKT3 resulted in comparable cloning efficiencies and clone sizes. Human serum was essential for high cloning efficiency as well as for continued growth, and could not be substituted with an increased amount of fetal calf serum. When cloning with interleukin-2 at 20,000 U/ml, two growing cell types were identified. The majority of the clones contained CD3+, CD4+, or CD8+ T cells. Ten out of 60 cultures however, contained cells with the CD3-16/56+ NK cell phenotype, indicating that the culture conditions stimulated proliferation of two different cell types. The described method can be applied for rational analysis of large numbers of minute cell cultures, in for example evaluation of different cloning conditions and estimation of precursor cell frequencies in limiting dilution analysis. The simultaneous phenotyping allows precursor cell analysis under conditions that stimulate growth of more than one cell type.     

Flow cytometry of v-Abl transformed pre-B cells heterogeneous in ectopic expression levels reveals Ras dose-response

Flow cytometry is a powerful tool for quantitative biology because it can perform single-cell analysis of large cell populations using multiple parameters. Results are often visualized as a two-dimensional scatter or contour plot. Because these plots can be relatively diffuse, it is not always straightforward to discern a relationship between measured parameters. We have demonstrated that quantitative trends can be fit to the single-cell data generated from a heterogeneous population. We engineered Abelson virus-transformed pre-B cells to express a broad range of oncogenic Ras levels. Instead of individual cultures with individual expression levels, a continuous range of levels was expressed by different cells in one heterogeneous culture. We then stained cells for downstream Erk phosphorylation to monitor MAPK signaling or employed an E2F-responsive genetic reporter to monitor cell-cycle activity. Subsequent analysis by flow cytometry and locally weighted scatterplot smoothing (LOWESS) revealed that increasing Ras oncogene expression led to increasing MAPK signaling. In contrast, E2F activity peaked at an optimal, intermediate level of Ras. To make this analytical method widely available to others, we have provided a software application that performs LOWESS on any two-parameter population data collected by flow cytometry.     

The use of flow cytometry in the diagnosis and monitoring of malignant hematological disorders

Flow cytometry is a modality with ever increasing application in modern hematological practice. This is due to the rapidity of obtaining results, ease of use and increasing power to detect abnormal populations of cells. The major uses of flow cytometry in malignant hematology are in the diagnosis, classification and monitoring of diseases such as leukemia, lymphoma and myeloma. The technique is now used also to detect disease-specific populations of cells in paroxysmal nocturnal hemoglobinuria. This review describes the use of flow cytometry in many disease states.     

High level production of soluble single chain antibodies in small-scale Escherichia coli cultures

We have investigated the effect of growth and induction conditions on the production of soluble single-chain Fv antibody fragments in Escherichia coli under the control of wt lac promoter. The scFv was directed into the periplasmic space by a pelB leader sequence. Addition of sucrose to the medium gave a 15–25-fold increase in the yield of soluble scFv-phOx (3.0 mg/l) for bacterial shake-tube cultures and an increase of 80–150-fold (16.5 mg/l) for shake-flask cultures. Using flask culture in the presence of 0.4 M sucrose, a significant amount of scFv was released into the medium. We found that the scFv could be made to accumulate in the periplasm or be secreted into the medium by simply changing the incubation conditions and the concentration of the inducer. The ratio between soluble antibody fragments and insoluble scFv aggregates proved to be dependent on the strength of the promoter. Lowering the incubation temperature below 20°C had no effect on the yield of soluble antibody fragments in the periplasm, but they were no longer secreted into the medium. An example of high level production in shake-flask cultures and one-step purification by immobilized metal affinity chromatography (IMAC) is described for a soluble scFv specific for the T cell surface antigen CD3. The biological activity of the purified anti-CD3 scFv was demonstrated by flow cytometry. This method should be especially useful for the functional screening of a large number of clones in small-scale cultures.     

Stimulation of the differentiation of osteogenic rat bone marrow stromal cells by osteoblast cultures

The effect of conditioned medium (CM) from rat calvaria (RC) cel cultures on the growth and differentiation of osteogenic cells in rat bone marrow stromal cell (BMSC) cultures was investigated. Control cultures received either CM from periodontal ligament fibroblast cultures or fresh medium. RCCM stimulated the formation of nodules of bonelike tissue in bone marrow stromal cell cultures in a dose-dependent manner,and the maximal stimulation was associated with the osteoblast-enriched cell populations of the RC cultures. Ultrafiltration demonstrated that activity was confined to a CM fraction of 10- to 30-kilodalton molecular size. The activity was sensitive to boiling and trypsin treatments, but was not affected by neutralizing antibodies to transforming growth factor beta or insulin-like growth factor I or II. RCCM was found to initially increase the number and proportion of cells that expressed alkaline phosphatase activity, although the proportion of alkaline phosphatase-positive cells subsequently declined. These data were consistent with an initial stimulation of proliferation of a subpopulation of osteoprogenitor cells within the cultures, followed by their differentiation. The results suggest that mature osteoblasts may produce a paracrine growth factor that can stimulate the differentiation of osteoblasts from precursor cells.     

Evidence for genetic predisposition for some nasopharyngeal cancers by in vitro hyperdiploidy in human dermal fibroblasts

A numerical alteration in chromosome complement in human dermal fibroblast cultures, hyperdiploidy with a normal occurrence of tetraploidy (IVH), has been reported to be associated with some hereditary single tumors including squamous carcinoma of the nasopharynx (NPC). Its incidence was compared in cultures derived from 39 NPC patients and 29 clinically normal subjects without a family cancer history by two different methods (percentage of numerically altered metaphases in chromosome preparations from nonconfluent monolayer Petri dish cultures in logarithmic growth, and by the distribution of DNA content of propidium iodide stained cells from plastic flask cultures in stationary growth phase as assayed by flow cytometry) to ascertain its usefulness in identification of such genetic predisposition. Concordance was observed between the two assays. There was a linear relationship between the percentage of hyperdiploid metaphases assayed in the chromosome preparations and the percentage of cells with a DNA index of greater than 1 as determined by flow cytometry. By metaphase assay, none of the 29 normals showed IVH. OF the 39 carcinoma of the nasopharynx patients studied, 19 had IVH and 20 did not. By flow cytometry there were significant differences in the flow cytometry DNA index (p less than 0.001) of IVH-negative (all normals and 20 carcinoma of the nasopharynx patients) and IVH-positive carcinoma of the nasopharynx patients. The percentage of cells with a DNA index of 2 and greater than 1 could be used to distinguish all IVH-negative from the IVH-positive subjects and, thus, were considered to be the parameters of choice in assaying IVH by flow cytometry. None of the subjects studied showed increased in vitro tetraploidy (IVT), which has been associated with some heritable colon cancer syndromes. Irrespective of family cancer history, approximately one-half of the NPC patients (19 of 39) had IVH, which has been reported to be associated with the in vivo expression of certain heritable tumors including carcinoma of the nasopharynx. The average age of carcinoma of the nasopharynx diagnosis was earlier (mean 52 yr) for the IVH-positive group than for the IVH-negative carcinoma of the nasopharynx group (mean 67 yr).     

Use of hydroethidine and flow cytometry to assess the effects of leukocytes on the malarial parasite Plasmodium falciparum.

Flow cytometry was evaluated as a method of assessing in vitro the effects of leukocytes on blood-stage Plasmodium falciparum. Hydroethidine is converted by metabolizing cells to ethidium, a nucleic acid fluorochrome. After incubation with hydroethidine, viable and dead leukocytes and parasitized and uninfected erthrocytes could all be identified on the basis of fluorescence intensity and size. Leukocytes can therefore be eliminated from further analysis; this allows assessment, at any parasite developmental stage, of the level of parasitemia within erythrocytes in the presence of any of several types of leukocytes. Whether leukocytes actually kill intraerythrocytic parasites can therefore be determined and the level of cytotoxicity can be assessed. The ability of leukocytes to prevent merozoites from invading new erythrocytes, i.e., inhibition of parasite invasion, can also be assessed by this method. When erythrocytes containing schizont-stage parasites were cocultured with different leukocyte populations and the level of parasitemia was determined after merozoite release and invasion, only cultures containing gamma delta T cells inhibited parasite invasion. The different blood-stage forms of the parasite vary in nucleic acid content, which allows each of the developmental stages to be distinguished by flow cytometry; this permits assessment of changes in parasite development in the presence of leukocytes. Monocyte-derived macrophages (MDMs) appeared to have an effect on parasite development. In this instance, when erythrocytes containing ring-form parasites were cocultured with MDMs and harvested 24 h later, the parasites in cultures containing MDMs were at the late schizont stage, whereas parasites in control cultures were early trophozoites; this finding suggests that MDMs accelerate parasite development. Together, these results indicate that flow cytometry is potentially useful for measuring the following effects mediated by leukocytes: (i) level of cytotoxicity, (ii) changes in parasite development, and (iii) inhibition of parasite invasion.     

Sucrose to polycose preference shifts in rats: the role of taste, osmolality and the fructose moiety

Rats given continuous access to 32% sucrose and Polycose solutions initially preferred sucrose but over days reversed their preference and drank more Polycose than sucrose. The role of taste and postingestive factors in this preference shift were investigated in several experiments. A strong and persistent preference for saccharin-sweetened Polycose over unsweetened Polycose was eliminated by intervening experience with a sucrose vs. Polycose choice test, suggesting that the postingestive effects modify the attractiveness of sweet taste. Osmolality differences between sucrose and Polycose were ruled out as an important factor; increasing the osmolality of Polycose by addition of glucose did not prevent the sucrose to Polycose preference shift. Rather, the shift in preference appears to result from some postingestive consequence of the fructose moiety contained in sucrose. This is indicated by the findings that rats did not reliably prefer a fructose-Polycose mixture to a sucrose solution in long-term tests, but developed preferences for maltose over sucrose solutions, and for glucose + Polycose over fructose + Polycose mixtures. Also, a 32% glucose solution was strongly preferred to a 32% fructose solution. Other studies examined the parameters of the sucrose to Polycose preference shift. The Polycose preference was accomplished by increases in both Polycose meal size and frequency. It occurred only with relatively concentrated solutions (16 and 32%) and was incomplete when the carbohydrates were presented in powdered form. Preexposure to Polycose enhanced the preference shift, whereas preexposure to sucrose retarded the development of the preference, suggesting that the shift occurs because Polycose is more rewarding, rather than because sucrose is more aversive. Polycose preference was lost after several days without the solutions but redeveloped in subsequent sucrose vs. Polycose choice tests. Thus the preference shift appears to be due to taste-postingestive consequence conditioning but its expression is dependent upon the nutritional state of the animal.     

Blood cells of sea bass (Dicentrarchus labrax L.). Flow cytometric and microscopic studies

Studies of fish blood cells made to date presented numerous problems derived from both the nomenclature and the techniques used. A combination of quantitative and morphological methods is needed if the classification of fish blood cells is to advance from it present provisional state. The aim of the present paper was first to isolate sea bass blood cell populations by flow cytometry and second to characterize then microscopically. Blood cell populations from sea bass (Dicentrarchus labrax L.) were isolated according to their FSC (size) and SSC (granularity) properties by flow cytometry. The isolated populations were then processed for light and transmission and scanning electron microscopic characterization. Sea bass blood leukocytes isolated by flow cytometry consisted of two main cell subpopulations. Subsequent microscopic study of these cells revealed that the first subpopulation was composed of small cells (3-5 microm) of low granularity and consisted of thrombocytes and lymphocytes whereas, the second subpopulation was formed of 6-9 microm sized cells of high granularity consisting of granulocytes and monocyte/macrophages. The combined use of flow cytometry and electron microscopy makes it possible to characterize the different cell types present in sea bass peripheral blood with a high degree of certainty. Although sea bass basically follows the common vertebrate hematological pattern, significant modifications such as the presence of circulating immature erythrocytes, plasma cells and monocyte/macrophages and different forms of thrombocytes can be established with respect to this pattern.     

Degradation of Levan by Actinomyces viscosus

Actinomyces viscosus ATCC 15987 was examined for its ability to hydrolyze its own levan. Washed whole cells and an ammonium sulfate fraction from cell-free culture fluids were shown to possess levan hydrolase activity. Analyses of reaction mixtures by gel filtration and thin-layer chromatography demonstrated that the product of levan hydrolysis was free fructose. The cell-associated and extracellular enzyme preparations also hydrolyzed inulin and the levans synthesized by Aerobacter levanicum and Bacillus subtilis. Growth of A. viscosus in media supplemented with 0.1% A. viscosus levan resulted in a 33-fold increase and a 7-fold increase in the specific activities of the respective extracellular and cell-associated enzymes when compared with those from 55 mM glucose cultures. Growth in the presence of 29.2 mM sucrose resulted in a 28-fold increase and a 5-fold increase in the specific activities of the respective enzymes when compared with those from the glucose cultures. The extracellular enzyme exhibited high activity over a wide pH range, with 87 and 89% of its pH 6.0 optimum activity at pH 5.0 and 7.0, respectively. The cell-associated enzyme also exhibited optimum activity at pH 6.0, but this was decreased to 10 and 20% at pH 5.0 and 7.0, respectively. Analysis for the presence of extracellular levan during growth of A. viscosus in sucrose broths demonstrated that peak levan concentrations occurred during the mid-exponential to late-exponential phase of growth followed by a rapid decline in extracellular levan as a result of levan hydrolase activity.     

Double fluorescence analysis of human cytomegalovirus (HCMV) infected human fibroblast cultures by flow cytometry: increase of class I MHC expression on uninfected cells and decrease on infected cells

Summary Cultured human foreskin fibroblasts (HFF) were infected with different multiplicities of infection (moi 0.001-0.1) of human cytomegalovirus (HCMV) strain AD 169 or a clinical isolate. Percentage of infected cells was determined by analysis of immediate early (IEA), early (EA), and late (LA) virus antigen expression with flow cytometry or by immunoperoxidase staining. Changes in the expression of class I MHC surface molecules were demonstrated by comparing the mean fluorescence intensities of infected HFF cultures with those of mock infected cell cultures by flow cytometry. At day three post infection single fluorescence analysis showed that infected HFF cultures split into low and high density class I MHC bearing cells. The addition of anti-interferon reduced the expression of class I MHC, distinctly. The assumption that infected cells down-regulate and uninfected cells up-regulate their expression of class I MHC molecules was demonstrated by double fluorescence analysis both with flow cytometry and fluorescence microscopy. Analysis of class I MHC-antigen expression versus immediate (IEA, mab E13), early (EA, mab 9221), or late (LA, mab BM219) virus antigen expression yielded three cell populations of HCMV infected HFF cultures three days post infection: 1. uninfected cells with an increase of class I MHC, 2. high density class I MHC, IEA and/or EA expressing cells, and 3. low class I MHC, IEA, EA and LA expressing cells.