Method of and apparatus for detecting microorganisms

• US 5,480,804 A
• Filed: 04/13/1993
• Issued: 01/02/1996
• Est. Priority Date: 06/28/1989
• Status: Expired due to Term

• Current Assignee: Kirin Beverage Corporation (Kirin Holdings Company Limited)
• Original Assignee: Kirin Beverage Corporation (Kirin Holdings Company Limited)
• Inventors: Ode, Takahiro, Yasui, Tetsuji, Niwa, Motohiro
• Primary Examiner(s): Beisner, William H.
• Application Number:
  US08/047,606
• Time in Patent Office: 994 Days
• Field of Search: 435/29, 435/30, 435/34, 435/284, 435/291, 435/808, 422/82.05, 422/82.08, 356/36, 356/38, 356/337, 356/338, 356/341, 356/343, 250/458.1-462.1
• US Class Current: 435/286.1
• CPC Class Codes: C12M 41/46 of cellular or enzymatic activity or functionality, e.g. cell viability
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6456 Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: Y10S 435/808 Optical sensing apparatus

Abstract

A microorganism detecting apparatus comprising a fluorescence microscope section which is furnished with a motor-driven stage for placing thereon a microorganism sample subjected to fluorescent staining, and a light source portion for projecting excitation light of predetermined wavelength on the sample; a detection section which detects and photoelectrically converts fluorescence of specified wavelength from the microorganism sample at a position posterior to the microscope section; a filter unit which limits the band of frequencies ascribable to noise in relation to an electric signal from the detection section; a signal processor which reads the output value of the band-limited signal from the detection section, and which processes the read output value; and an automatic inspection section which drives the stage so as to permit the microorganism sample to be scanned over its whole area, and which stores each signal detection part in the sample so as to permit a fluorescent substance at the part to be verified.

Claims

1. An apparatus for detecting fluorescent substances in a microorganism sample, the apparatus comprising:

stage means for placing thereon a microorganism sample subjected to fluorescent staining, and for transporting the sample to permit the same to be scanned;

light source means for protecting excitation light of a predetermined wavelength onto the sample so that a fluorescent light is originated from the sample;

slit means having a slit of a predetermined width through which the fluorescent light from the sample passes;

sensor means for sensing the fluorescent light which has passed through the slit to obtain an electric signal;

signal filter means for limiting a frequency band of the electric signal from the sensor means to a frequency band predetermined depending on a width of the slit as imaged onto the sample and a speed of the scanning of the sample; and

signal processing means for processing the band-limited electric signal so as to detect fluorescent substances in the sample,

wherein the sample includes a plurality of colonies of microorganisms having diameters in the range of D₁ to D₂ and the frequency band of the electric signal is selected to obtain electric signals at a frequency R that corresponds to the diameter of an individual colony, where

space="preserve" listing-type="equation">R=S/(W+D) (Hz)

wherein S is the speed of the scanning of the sample,

W is the width of the slit as imaged onto the sample, and

D is the diameter of a colony being scanned, and

the frequency range extends at least from a frequency R₁ for colonies of diameter D₁ to a frequency R₂ for colonies of diameter D₂.

2. An apparatus according to claim 1, wherein said sensor means includes a photomultiplier tube for the photoelectric conversion of the fluorescent light.

3. An apparatus according to claim 1, wherein the microorganism sample is a sample in which the microorganisms in an object to-be-inspected are caught on a membrane filter and then subjected to the fluorescent staining.

4. An apparatus for detecting microorganisms in a microorganism sample subjected to fluorescent staining, comprising:

stage means for placing thereon a microorganism sample subjected to fluorescent staining, and for transporting the sample to permit the sample to be scanned;

light source means for projecting an excitation light of a predetermined wavelength onto the sample so that a fluorescent light is originated from the sample;

slit means having a slit of a predetermined width through which the fluorescent light from the sample passes;

first optical filter means for filtering the fluorescent light from the sample to obtain a first band-limited light of a first wavelength band;

second optical filter means for filtering the fluorescent light from the sample to obtain a second hand, limited light of a second wavelength;

first sensor means for sensing the first band-limited light which has passed through the slit to obtain a first electric signal;

second sensor means for sensing the second band-limited light which has passed through the slit to obtain a second electric signal;

signal filter means for limiting a frequency band of the electric signals from the sensor means to a frequency band predetermined depending on a width of the slit as imaged onto the sample and a speed of the scanning of the sample; and

processing means for processing the band-limited first and second electric signals to discriminate the microorganisms from auto-fluorescent foreign matter in the sample by utilizing a discrepancy between fluorescence spectra from the microorganisms and the auto-fluorescent foreign matter,

wherein the sample includes a plurality of colonies of microorganisms having diameters in the range of D₁ to D₂, and the frequency band of the electric signals is selected to obtain electric signals at a frequency R that corresponds to the diameter of an individual colony, wherein

space="preserve" listing-type="equation">R=S/(W+D) (Hz)

wherein S is the speed of the scanning of the sample,

W is the width of the slit as imaged onto the sample, and

D is the diameter of a colony being scanned, and

the frequency range extends at least from a frequency R₁ for colonies of diameter D1 to a frequency R₂ for colonies of diameter D₂.

5. An apparatus for detecting microorganisms according to claim 4, wherein the microorganisms in the sample are caught on a membrane filter and then subjected to the fluorescent staining.

6. An apparatus for detecting microorganisms according to claim 4, wherein said first and second sensor means include a photomultiplier tube for the photoelectric conversion of the fluorescent light.

7. An apparatus for detecting microorganisms according to claim 4, wherein B excitation light having a wavelength of about 490 nm is employed as the excitation light, and two sorts of optical filters whose pass bands are respectively of 500-600 nm and 550-700 nm are employed as said first and second optical filter means.

8. An apparatus for detecting microorganisms according to claim 4, wherein V excitation light having a wavelength of about 405 nm are employed as the excitation light, and two sorts of optical filters whose pass bands are respectively at 480-530 nm and 510-560 nm are employed as said first and second optical filter means.